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.

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.

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.

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.

Three-dimensional porous poly(propylene fumarate)-co-poly(lactic-co-glycolic acid) scaffolds for tissue engineering.

Three-dimensional structural scaffolds have played an important role in tissue engineering, especially broad applications in areas such as regenerative medicine. We have developed novel biodegradable porous poly(propylene fumarate)-co-poly(lactic-co-glycolic acid) (PPF-co-PLGA) scaffolds using thermally induced phase separation, and determined the effects of critical parameters such as copolymer concentration (6, 8, and 10 wt %) and the binary solvent ratio of dioxane:water (78/22, 80/20, 82/18 wt/wt %) on the fabrication process. The cloud-point temperatures of PPF-co-PLGA changed in parallel with increasing copolymer concentration, but inversely with increasing dioxane content. The compressive moduli of the scaffolds increased with greater weight composition and dioxane:water ratio. Scaffolds formed using high copolymer concentrations and solvent ratios exhibited preferable biomineralization. All samples showed biodegradation capability in both accelerated solution and phosphate-buffered saline (PBS). Cell toxicity testing indicated that the scaffolds had good biocompatibility with bone and nerve cells, which adhered well to the scaffolds. Variations in the copolymer concentration and solvent ratio exercised a remarkable influence on morphology, mechanical properties, biomineralization, and biodegradation, but not on the cell viability and adhesion of the cross-linked scaffolds. An 8 to 10 wt % solute concentration and 80/20 to 82/18 wt/wt dioxane:water ratio were the optimum parameters for scaffold fabrication. PPF-co-PLGA scaffolds thus possess several promising prospects for tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2507-2517, 2018.

Angiogenic Rg1 /Sr-Doped TiO2 Nanowire/Poly(Propylene Fumarate) Bone Cement Composites.

A new approach is provided for preparing radiopaque and angiogenic poly(propylene fumarate) (PPF) bone cements by integrating Sr-doped n-TiO2 nanowires and ginsenoside Rg1 suitable for treating osteonecrosis. High aspect ratio radiopaque TiO2 -nanowires are synthesized by strontium doping in supercritical CO2 for the first time, showing a new phase, SrTiO3 . PPF is synthesized using a transesterification method by reacting diethyl fumarate and propylene glycol, then functionalized using maleic anhydride to produce terminal carboxyl groups, which are subsequently linked to the nanowires. The strong interfacial adhesion between functionalized PPF and nanowires is examined by scanning electron microscopy, Fourier transform infrared, X-ray photoelectron spectroscopy, thermal analysis, and mechanical testing. An angiogenic modulator, ginsenoside Rg1 , is integrated into the bone cement formulation with the mechanical properties, radiopacity, drug release, and angiogenesis behavior of the formed composites explored. The results show superior radiopacity and excellent release of ginsenoside Rg1 in vitro, as well as a dose-dependent increase in the branching point numbers. The present study suggests this new methodology provides sufficient mechanical properties, radiopacity, and angiogenic activity to be suitable for cementation of necrotic bone.

Controlled Delivery of Vancomycin via Charged Hydrogels.

Surgical site infection (SSI) remains a significant risk for any clean orthopedic surgical procedure. Complications resulting from an SSI often require a second surgery and lengthen patient recovery time. The efficacy of antimicrobial agents delivered to combat SSI is diminished by systemic toxicity, bacterial resistance, and patient compliance to dosing schedules. We submit that development of localized, controlled release formulations for antimicrobial compounds would improve the effectiveness of prophylactic surgical wound antibiotic treatment while decreasing systemic side effects. Our research group developed and characterized oligo(poly(ethylene glycol)fumarate)/sodium methacrylate (OPF/SMA) charged copolymers as biocompatible hydrogel matrices. Here, we report the engineering of this copolymer for use as an antibiotic delivery vehicle in surgical applications. We demonstrate that these hydrogels can be efficiently loaded with vancomycin (over 500 µg drug per mg hydrogel) and this loading mechanism is both time- and charge-dependent. Vancomycin release kinetics are shown to be dependent on copolymer negative charge. In the first 6 hours, we achieved as low as 33.7% release. In the first 24 hours, under 80% of total loaded drug was released. Further, vancomycin release from this system can be extended past four days. Finally, we show that the antimicrobial activity of released vancomycin is equivalent to stock vancomycin in inhibiting the growth of colonies of a clinically derived strain of methicillin-resistant Staphylococcus aureus. In summary, our work demonstrates that OPF/SMA hydrogels are appropriate candidates to deliver local antibiotic therapy for prophylaxis of surgical site infection.

In vivo tumor-targeted dual-modal fluorescence/CT imaging using a nanoprobe co-loaded with an aggregation-induced emission dye and gold nanoparticles.

As an intensely studied computed tomography (CT) contrast agent, gold nanoparticle has been suggested to be combined with fluorescence imaging modality to offset the low sensitivity of CT. However, the strong quenching of gold nanoparticle on fluorescent dyes requires complicated design and shielding to overcome. Herein, we report a unique nanoprobe (M-NPAPF-Au) co-loading an aggregation-induced emission (AIE) red dye and gold nanoparticles into DSPE-PEG(2000) micelles for dual-modal fluorescence/CT imaging. The nanoprobe was prepared based on a facile method of "one-pot ultrasonic emulsification". Surprisingly, in the micelles system, fluorescence dye (NPAPF) efficiently overcame the strong fluorescence quenching of shielding-free gold nanoparticles and retained the crucial AIE feature. In vivo studies demonstrated the nanoprobe had superior tumor-targeting ability, excellent fluorescence and CT imaging effects. The totality of present studies clearly indicates the significant potential application of M-NPAPF-Au as a dual-modal non-invasive fluorescence/X-ray CT nanoprobe for in vivo tumor-targeted imaging and diagnosis.

Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging.

We report a new strategy of using carrier-free pure near-infrared (NIR) dye nanoparticles (NPs) to achieve highly luminescent NIR fluorescent probes for in vitro and in vivo imaging. Bis(4-(N-(2-naphthyl)phenylamino) phenyl)-fumaronitrile (NPAPF) NPs are shown to exhibit favorable biocompatibility, wide-range pH stability (pH 4-10) and much more superior photostability than conventional dyes. Importantly, the combined merits of high dye loading content and aggregation-induced emission enhancement properties, endow the NIR probes with high brightness and a high quantum yield up to 14.9%. The NPAPF NPs can be readily conjugated with folic acid for targeted in vitro cell imaging. Applications of the NPs probes in high efficiency in vivo and ex vivo imaging were successfully demonstrated. Intense fluorescent signals of NPAPF NPs can be distinctly, selectively and spatially resolved in tumor sites with ultrahigh sensitivity, even with 5 ms exposure time, due to the preferentially accumulation of NPs in tumor sites through passive enhanced permeability and retention effect. The totality of results clearly demonstrate the exciting potential of the functionalized NPAPF NPs as a NIR fluorescent probe for in vitro and in vivo imaging and diagnostics.

Synthesis of oligo(poly(ethylene glycol) fumarate).

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.

Discovery of a novel pyrrole derivative 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate (TAK-438) as a potassium-competitive acid blocker (P-CAB).

In our pursuit of developing a novel and potent potassium-competitive acid blocker (P-CAB), we synthesized pyrrole derivatives focusing on compounds with low log D and high ligand-lipophilicity efficiency (LLE) values. Among the compounds synthesized, the compound 13e exhibited potent H(+),K(+)-ATPase inhibitory activity and potent gastric acid secretion inhibitory action in vivo. Its maximum efficacy was more potent and its duration of action was much longer than those of proton pump inhibitors (PPIs). Therefore, compound 13e (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate, TAK-438) was selected as a drug candidate for the treatment of gastroesophageal reflux disease (GERD), peptic ulcer, and other acid-related diseases.

[Effect of intragastric creatine glycine ethylic ether fumarate administration in a rat model of occlusive ischemia].

The aim of the study was to investigate neuroprotective effect of creatine glycine ethylic ether fumarate (creamide). The methods involved intragastric administration of creamide in doses of 30 and 50 mg/kg twice a day for 10 days. Focal 30 minutes cerebral ischemia model by endovascular suture occlusion of the middle cerebral artery in a rat with subsequent reperfusion period for 48 hours was produced. Assessment of creamide stability in gastric juice was performed. Ischemic lesion volume accompanying focal ischemia was visualized and determined. Similar infarction patterns had been found with histological methods. Garcia scale was used for clinical study of neurological deficit in rats. Our data suggest a significant neuroprotective effect of creamide in dosage 50 mg/kg administered twice a day which decreased brain lesion volume produced by ischemic and reperfusion injury.

Hydroxyapatite scaffolds infiltrated with thermally crosslinked polycaprolactone fumarate and polycaprolactone itaconate.

In this work, two unsaturated derivatives of polycaprolactone (PCL), polycaprolactone fumarate (PCLF), and polycaprolactone itaconate (PCLI), have been synthesized and used as an infiltrating polymer to improve the mechanical properties of brittle hydroxyapatite (HA) scaffolds. PCLF and PCLI were first synthesized through polyesterification of the low molecular weight PCL diols with fumaryl chloride and itaconyl chloride respectively, and then characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, gel permeation chromatography, and differential scanning calorimetry analysis. HA scaffolds were sintered using a foam replication technique, with porosity of about 60%. Polymer-HA composites were obtained by infiltrating the HA scaffolds with PCLF and PCLI solution (12.5 and 30 w/v in dichloromethane) followed by thermal crosslinking. The polymer infiltrated HA scaffolds were characterized by scanning electron microscopy, porosimetry, and gravimetrical analysis. The polyesterification reaction of PCL diols with fumarate chloride was more efficient than itaconyl chloride and dependent upon the molecular weight of the initial PCL precursor; the resultant PCLF demonstrated a degree of substitution of 1.2, 4.2, and 2.7 times higher than PCLIs. Polymer infiltration improved the compressive strength of the HA scaffolds, and based upon the type of macromer (PCLF or PCLI) and also their concentration in infiltrating solution (12.5 or 30 w/v %) compressive strength increased about 14-328%. In all studied samples, the reinforcement effect of PCLF infiltration was higher than PCLI. The macromers and their corresponding infiltrated HA scaffolds did not show any significant cytotoxicity toward human primary osteogenic sarcoma cell (G92 cell lines), in vitro.

Cross-linking characteristics and mechanical properties of an injectable biomaterial composed of polypropylene fumarate and polycaprolactone co-polymer.

In this work, a series of co-polymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight and PCL fraction in the co-polymer (PCL feed ratio) on the maximum cross-linking temperature, gelation time and mechanical properties of the cross-linked co-polymers were investigated. The maximum cross-linking temperature fell between 38.2 ± 0.3 and 47.2 ± 0.4°C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2 ± 0.2 and 8.5 ± 0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44 ± 1.8 to 142 ± 7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1 ± 0.3 and 17.1 ± 1.3 kJ/m(3). Our data suggest that the cross-linking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore, the PPF-co-PCL co-polymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.

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.

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.

On-bead screening of a combinatorial fumaric acid derived peptide library yields antiplasmodial cysteine protease inhibitors with unusual peptide sequences.

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K(i) values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.

Synthesis of poly(propylene fumarate).

This protocol describes the synthesis of 500-4,000 Da poly(propylene fumarate) (PPF) by a two-step reaction of diethyl fumarate and propylene glycol through a bis(hydroxypropyl) fumarate diester intermediate. Purified PPF can be covalently cross-linked to form degradable polymer networks, which have been widely explored for biomedical applications. The properties of cross-linked PPF networks depend upon the molecular properties of the constituent polymer, such as the molecular weight. The purity of the reactants and the exclusion of water from the reaction system are of utmost importance in the generation of high-molecular-weight PPF products. Additionally, the reaction time and temperature influence the molecular weight of the PPF product. The expected time required to complete this protocol is 3 d.

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.