Injectable Catalyst-Free Poly(Propylene Fumarate) System Cross-Linked by Strain Promoted Alkyne-Azide Cycloaddition Click Chemistry for Spine Defect Filling.

A new PPF-BCN/hyPCL32-N3 injectable system that can be cross-linked by catalyst-free, strain promoted alkyne-azide cycloaddition (SPAAC) click chemistry was developed for tissue engineering applications. The system consisted of two components: PPF-BCN, poly(propylene fumarate) (PPF) functionalized with (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH), and hyPCL32-N3, a hyper-branched 32-arm poly(ε-caprolactone) (PCL) dendrimer functionalized with azide as the cross-linker core. Fast SPAAC click reaction allowed the desired gelation of the system without using any toxic initiator or catalyst. Compared to the conventional injectable formulation, e.g., poly(methyl methacrylate) (PMMA), our PPF-BCN/hyPCL32-N3 (abbreviated as PFCL-Click) injectable system showed enhanced biocompatibility and low heat generation during cross-linking. After reaction, the cross-linked PFCL-Click scaffolds supported excellent proliferation and differentiation of preosteoblast cells on the surface. The PFCL-Click system can be successfully injected into vertebral bodies of rabbit spine and can be monitored by X-ray imaging after incorporating zirconium dioxide (ZrO2) powder. With these unique advantages, this injectable system has promising potential for bone defect repair and other tissue engineering and regenerative medicine applications.

Ring-Opening Copolymerization of Maleic Anhydride with Functional Epoxides: Poly(propylene fumarate) Analogues Capable of Post-Polymerization Modification.

Three functional epoxides were copolymerized with maleic anhydride to yield degradable poly(propylene fumarate) analogues. The polymers were modified post-polymerization and post-printing with either click-type addition reactions or UV deprotection to either attach bioactive species or increase the hydrophilicity. Successful dye attachment, induced wettability, and improved cell spreading show the viability of these analogues in biomaterials applications.

Synthesis and Characterization of Diol-Based Unsaturated Polyesters: Poly(diol fumarate) and Poly(diol fumarate-co-succinate).

In this work, we describe the synthesis and characterization of variants of poly(diol fumarate) and poly(diol fumarate-co-succinate). Through a Fischer esterification, α,ω-diols and dicarboxylic acids were polymerized to form aliphatic polyester comacromers. Because of the carbon-carbon double bond of fumaric acid, incorporating it into the macromer backbone structure resulted in unsaturated chains. By choosing α,ω-diols of different lengths (1,6-hexanediol, 1,8-octanediol, and 1,10-decanediol) and controlling the amount of fumaric acid in the dicarboxylic acid monomer feed (33, 50, and 100 mol %), nine diol-based macromer variants were synthesized and characterized for molecular weight, number of unsaturated bonds per chain, and thermal properties. Degradation and in vitro cytotoxicity were also measured in a subset of macromers. As proof-of-principle, macromer networks were photo-cross-linked to demonstrate the ability to perform free radical addition using the unsaturated macromer backbone. Cross-linked macromer networks were also characterized for physicochemical properties (swelling, sol fraction, compressive modulus) based on diol length and amount of unsaturated bonds. A statistical model was built using data generated from these diol-based macromers and macromer networks to evaluate the impact of monomer inputs on final macromer and macromer network properties. With the ability to be modified by free radical addition, biodegradable unsaturated polyesters serve as important macromers in the design of devices such as drug delivery vehicles and tissue scaffolds. Given the ability to extensively control final macromer properties based on monomer input parameters, poly(diol fumarate) and poly(diol fumarate-co-succinate) represent an exciting new class of macromers.

Formal total synthesis of aliskiren.

The efficient and selective formal total synthesis of aliskiren is described. Aliskiren, a renin inhibitor drug, has received considerable attention, primarily because it is the first of the renin inhibitor drugs to be approved by the FDA. Herein, the formal synthesis of aliskiren by iridium-catalyzed asymmetric hydrogenation of two allylic alcohol fragments is reported. Screening a number of N,P-ligated iridium catalysts yielded two catalysts that gave the highest enantioselectivity in the hydrogenation, which gave the saturated alcohols in 97 and 93 % ee. In only four steps after hydrogenation, the fragments were combined by using the Julia-Kocienski reaction to produce late-stage intermediate in an overall yield of 18 %.

The development of a complementary pathway for the synthesis of aliskiren.

The synthesis of aliskiren (1), a recently marketed drug for the treatment of hypertension, is presented. The focus of our synthetic effort is to develop an efficient pathway for the synthesis of (2S,7R,E)-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy) benzyl)-N,N,8-trimethylnon-4-enamide (2a), which has been used as the advanced intermediate toward aliskiren. After an extensive investigation of three different strategies designed to construct the E-olefin functionality in 2a by employing the olefin cross-metathesis, Horner-Wadsworth-Emmons (HWE), and Julia-type olefinations, we have established a new protocol for the synthesis of 2a with a substantially improved overall efficiency in terms of the yield (ca. 33%), and diastereo- and E/Z-selectivity. The key transformations were the Evans chiral auxiliary-aided asymmetric allylation for the synthesis of the appropriate chiral intermediates in excellent enantiomeric purity of higher than 97% ee and a modified Julia-Kocienski olefination for the highly selective construction of E-2a with up to 13.6:1 E/Z ratio from the chiral intermediates. Consequently, the results provide an appealing option for the synthesis of aliskiren.

Conception and evolution of stereocontrolled strategies toward functionalized 8-aryloctanoic acids related to the total synthesis of aliskiren.

A detailed account is given describing the approaches used toward the total synthesis of aliskiren. In particular, ring-closing metathesis with the Hoveyda-Grubbs catalyst accelerates the formation of a 9-membered lactone from an (R)-ester. The diastereomeric (S)-ester leads to the formation of dimeric dilactones, which were characterized by X-ray analysis and chemical conversions.

Fumarate copolymers-based membranes overlooking future transdermal delivery devices: synthesis and properties.

Novel copolymers of vinyl acetate and dialkylfumarates, poly(VA-co-DRF) with R = isopropyl (DIPF) or octan-2-yl (DOF), were synthesized by radical copolymerization under microwave conditions. The products were characterized by (1)H NMR and FTIR spectroscopies, size exclusion chromatography and differential scanning calorimetry. Based on these copolymers three membranes supported on polyvinyl alcohol were prepared and their morphology, swelling and mechanical properties were studied. The swelling kinetic was analyzed and interpreted in light of the Fick transport model, showing that the water transport occurs through a non-Fickian diffusion mechanism. The results show that the membrane prepared of poly(VA-co-DOF) exhibited excellent properties as potential platform for transdermal delivery system: they exhibited good tensile strength, moderated swelling and form thin and transparent films.

End-to-end continuous manufacturing of pharmaceuticals: integrated synthesis, purification, and final dosage formation.

A series of tubes: The continuous manufacture of a finished drug product starting from chemical intermediates is reported. The continuous pilot-scale plant used a novel route that incorporated many advantages of continuous-flow processes to produce active pharmaceutical ingredients and the drug product in one integrated system.

Exploratory experiments on the chemistry of the "glyoxylate scenario": formation of ketosugars from dihydroxyfumarate.

In the context of a "glyoxylate scenario" of primordial metabolism, the reactions of dihydroxyfumarate (DHF) with reactive small molecule aldehydes (e.g., glyoxylate, formaldehyde, glycolaldehyde, and glyceraldehyde) in water were investigated and shown to form dihydroxyacetone, tetrulose, and the two pentuloses, with almost quantitative conversion. The practically clean and selective formation of ketoses in these reactions, with no detectable admixture of aldoses, stands in stark contrast to the formose reaction, where a complex mixture of linear and branched aldoses and ketoses are produced. These results suggest that the reaction of DHF with aldehydes could constitute a reasonable pathway for the formation of carbohydrates and allow for alternative potential prebiotic scenarios to the formose reaction to be considered.

Surface characteristics of biomaterials used for space maintenance in a mandibular defect: a pilot animal study.

PURPOSE: The purpose of the present study was to evaluate the effect of implant porosity on wound healing between solid and porous implants placed within a bony mandibular defect with intraoral exposure. MATERIALS AND METHODS: Solid poly(methyl methacrylate) (PMMA) implants similar to those used currently in clinical space maintenance applications in maxillofacial surgery were compared with poly(propylene fumarate) implants that contained a porous outer surface surrounding a solid core. A 10-mm diameter nonhealing bicortical defect with open communication into the oral cavity was created in the molar mandibular region of 12 adult male New Zealand white rabbits. Of the 12 rabbits, 6 received the hybrid poly(propylene fumarate) implants and 6 received the solid PMMA implants. At 12 weeks, the rabbit mandibles were harvested and sent for histologic staining and sectioning. RESULTS: Gross inspection and histologic examination showed all 6 poly(propylene fumarate) implants to be intact within the defect site at the termination of the study period, with 3 of the 6 specimens exhibiting a continuous circumferential soft tissue margin. In contrast, 5 of the 6 PMMA-implanted specimens were exposed intraorally with an incomplete cuff of soft tissue around the implant. One of the PMMA-implanted specimens exhibited complete extrusion and subsequent loss of the implant. Fisher's exact test was used to compare the occurrence of oral cavity wound healing between the 2 groups (P = .09). CONCLUSIONS: Although statistically significant differences between the 2 groups were not seen, our results have indicated that advantages might exist to using porous implants for space maintenance. Additional study is needed to evaluate these findings.

Synthesis and characterization of a photo-cross-linked bioactive polycaprolactone-based osteoconductive biocomposite.

In this study, a light cross-linkable biocomposite scaffold based on a photo-cross-linkable poly (propylene fumarate) (PPF)-co-polycaprolactone (PCL) tri-block copolymer was synthesized and characterized. The developed biodegradable scaffold was further modified with ß-tricalcium phosphate (ß-TCP) bioceramic for bone tissue engineering applications. The developed biocomposite was characterized using H nuclear magnetic resonance and Fourier transform infrared spectroscopy. Moreover, the bioceramic particle size distribution and morphology were evaluated using Brunauer-Emmett-Teller method, X-ray diffraction, and scanning electron microscopy. The mechanical properties and biodegradation of the scaffolds were also evaluated. Cytotoxicity and mineralization assays were performed to analyze the biocompatibility and bioactivity capacity of the developed biocomposite. The characterization data confirmed the development of a biodegradable and photo-cross-linkable PCL-based biocomposite reinforced with ß-TCP bioceramic. In vitro analyses demonstrated the biocompatibility and mineralization potential of the synthesized bioceramic. Altogether, the results of the present study suggest that the photo-cross-linkable PCL-PPF-PCL tri-block copolymer reinforced with ß-TCP is a promising biocomposite for bone tissue engineering applications. According to the results, this newly synthesized material has a proper chemical composition for further clinically-relevant studies in tissue engineering.

Preparation of Sterile Raw Material - Chicken Eggshells in the Process of their Transformation into Selected Calcium Salts.

BACKGROUND: The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry. METHODS: The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C. RESULTS AND DISCUSSION: It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors. CONCLUSION: Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

A covalent succinylcysteine-like intermediate in the enzyme-catalyzed transformation of maleate to fumarate by maleate isomerase.

Maleate isomerase (MI), a member of the Asp/Glu racemase superfamily, catalyzes cis-trans isomerization of the C2-C3 double bond in maleate to yield fumarate. Mutational studies, in conjunction with the structure of the C194A mutant of Nocardia farcinica MI cocrystallized with maleate, have revealed an unprecedented mode of catalysis for the superfamily in which the isomerization reaction is initiated by nucleophilic attack of cysteine at the double bond, yielding a covalent succinylcysteine-like intermediate.

[FTIR study on the synthesis of poly(propylene fumarate) and its copolymer].

Poly(propylene fumarate) (PPF) is one kind of linear biodegradable polyester and the unsaturated double bonds along its main chain can be crosslinked with other olefinic monomers to form three-dimensional networks, and the networks can support tissues. In the present paper, firstly, the intermediate oligomer-bis (2-hydroxypropyl) fumarate (PFP) was synthesized, and then the unsaturated linear polyester PPF was synthesized with the oligomer PFP through melting condensation process. Additionally, on the base of the process, the oligomer bis(2-hydroxypropyl) sebacate (PSP) was synthesized by similar method and then a kind of new copolymer named poly(propylene fumarate-co-propylene sebacate) [P(PF-co-PS)] that comprised bis(2-hydroxypropyl) sebacate segments was synthesized with PFP and PSP by melting condensation. During the synthesis process, the structures of bis(2-hdroxypropyl) fumarate, bis(2-hydroxypropyl) sebacate, PPF and P(PF-co-PS) were characterized by FTIR The results shows that with the polymerization going along, oligomer bis (2-hydroxypropyl) fumarate and bis(2-hydroxypropyl) sebacate converted to PPF or P(PF-co-PS) gradually.

Studies on biodegradation of crosslinked hydroxy terminated-poly(proplyene fumarate) and formation of scaffold for orthopedic applications.

Biodegradation of crosslinked-hydroxy terminated-poly(proplyene fumarate) (X-HTPPF) has been studied in simulated physiological media to assess the formation of porous scaffold structure for bone growth and remodeling in load bearing orthopedic applications. Variation in crosslink density and surface hydrophilicity of X-HTPPF are observed due to non-stoichiometric mass of reacting partners. These variations influence absorption of the medium and biodegradation during aging. Though the initial absorption of medium is relatively higher with the crosslinked polymer (PNVP1) having 63.6% HT-PPF and 36.4% comonomer n-vinyl pyrrolidone (NVP) during the initial period of aging, the weight loss due to subsequent degradation with time is relatively lesser. PNVP1 undergo slow degradation with formation of fibril structure on the surface. The present crosslinked material PNVP1 is a candidate for the load bearing orthopedic applications.

Injectable biomaterials for minimally invasive orthopedic treatments.

Biodegradable and injectable hydroxy terminated-poly propylene fumarate (HT-PPF) bone cement was developed. The injectable formulation consisting HT-PPF and comonomer, n-vinyl pyrrolidone, calcium phosphate filler, free radical catalyst, accelerator and radiopaque agent sets rapidly to hard mass with low exothermic temperature. The candidate bone cement attains mechanical strength more than the required compressive strength of 5 MPa and compressive modulus 50 MPa. The candidate bone cement resin elicits cell adhesion and cytoplasmic spreading of osteoblast cells. The cured bone cement does not induce intracutaneous irritation and skin sensitization. The candidate bone cement is tissue compatible without eliciting any adverse tissue reactions. The candidate bone cement is osteoconductive and inductive and allow osteointegration and bone remodeling. HT-PPF bone cement is candidate bone cement for minimally invasive radiological procedures for the treatment of bone diseases and spinal compression fractures.

Molecular Mass-Dependent Resorption and Bone Regeneration of 3D Printed PPF Scaffolds in a Critical-Sized Rat Cranial Defect Model.

The emergence of additive manufacturing has afforded the ability to fabricate intricate, high resolution, and patient-specific polymeric implants. However, the availability of biocompatible resins with tunable resorption profiles remains a significant hurdle to clinical translation. In this study, 3D scaffolds are fabricated via stereolithographic cDLP printing of poly(propylene fumarate) (PPF) and assessed for bone regeneration in a rat critical-sized cranial defect model. Scaffolds are printed with two different molecular mass resin formulations (1000 and 1900 Da) with narrow molecular mass distributions and implanted to determine if these polymer characteristics influence scaffold resorption and bone regeneration in vivo. X-ray microcomputed tomography (µ-CT) data reveal that at 4 weeks the lower molecular mass polymer degrades faster than the higher molecular mass PPF and thus more new bone is able to infiltrate the defect. However, at 12 weeks, the regenerated bone volume of the 1900 Da formulation is nearly equivalent to the lower molecular mass 1000 Da formulation. Significantly, lamellar bone bridges the defect at 12 weeks with both PPF formulations and there is no indication of an acute inflammatory response.

Photo-cross-linked hybrid polymer networks consisting of poly(propylene fumarate) and poly(caprolactone fumarate): controlled physical properties and regulated bone and nerve cell responses.

Aiming to achieve suitable polymeric biomaterials with controlled physical properties for hard and soft tissue replacements, we have developed a series of blends consisting of two photo-cross-linkable polymers: polypropylene fumarate (PPF) and polycaprolactone fumarate (PCLF). Physical properties of both un-cross-linked and UV cross-linked PPF/PCLF blends with PPF composition ranging from 0% to 100% have been investigated extensively. It has been found that the physical properties such as thermal, rheological, and mechanical properties could be modulated efficiently by varying the PPF composition in the blends. Thermal properties including glass transition temperature (T g) and melting temperature (T m) have been correlated with their rheological and mechanical properties. Surface characteristics such as surface morphology, hydrophilicity, and the capability of adsorbing serum protein from culture medium have also been examined for the cross-linked polymer and blend disks. For potential applications in bone and nerve tissue engineering, in vitro cell studies including cytotoxicity, cell adhesion, and proliferation on cross-linked disks with controlled physical properties have been performed using rat bone marrow stromal cells and SPL201 cells, respectively. In addition, the role of mechanical properties such as surface stiffness in modulating cell responses has been emphasized using this model blend system.

Photopolymerization and shrinkage kinetics of in situ crosslinkable N-vinyl-pyrrolidone/poly(epsilon-caprolactone fumarate) networks.

Biodegradable, injectable and in situ photocrosslinkable macromers based on fumaric acid and polycaprolactone (PCLF) were prepared and characterized by FTIR, 1HNMR, and 13CNMR spectroscopy. The multifunctional macromers dissolved in N-vinyl pyrollidone (NVP) were photopolymerized by visible light irradiation in the presence of camphorquinone as photoinitiator. The photocrosslinking reaction was monitored by measuring shrinkage strain and shrinkage strain rate. The degree of photopolymerization reaction i.e. degree of conversion (DC%) was traced using FTIR spectroscopy. A three level factorial design was developed to study the effects of initiator concentration, NVP concentration, and molecular weight of PCLF upon photocrosslinking characteristics including degree of conversion and shrinkage strain. Results revealed that although neat PCLF was photopolymerized, but it was putty like after 220 seconds of irradiation and showed a very low degree of conversion (29%). Adding about 20% NVP caused a dramatic increase in its degree of conversion (63.33%). Increasing NVP up to 50% resulted in a decrease in DC% because of lower reactivity of NVP and leaving more unreacted NVP monomers. Sol fraction studies supported these results indicating that at higher NVP concentration, most of NVP and PCLF have not undergone the crosslinking reaction, leading to 55% decrease in DC%. Shrinkage strain measurement also confirmed the FTIR results.

Co-production of fumaric acid and chitin from a nitrogen-rich lignocellulosic material - dairy manure - using a pelletized filamentous fungus Rhizopus oryzae ATCC 20344.

Fumaric acid is widely used as a food additive for flavor and preservation. Rhizopus oryzae ATCC 20344 is a fungus known for good fumaric acid production. It also has been reported that the fungal biomass has high chitin content. This study investigated the possibility of producing both fumaric acid and chitin via R. oryzae fermentation of dairy manure. Co-production of valuable bio-based chemicals such as fumaric acid and chitin could make the utilization of manure more efficient and more profitable. A three step fermentation process was developed which effectively utilized the nitrogen as well as the carbohydrate sources within the manure. These steps were: the culturing of pellet seed; biomass cultivation on liquid manure to produce both biomass and chitin; and fumaric acid production on the hydrolysate from the manure fiber. Under the identified optimal conditions, the fermentation system had a fumaric acid yield of 31%, and a biomass concentration of 11.5 g/L that contained 0.21 g chitin/g biomass.