Bioactive Polyurethane-Poly(ethylene Glycol) Diacrylate Hydrogels for Applications in Tissue Engineering.

Polyurethanes (PUs) are a highly adaptable class of biomaterials that are among some of the most researched materials for various biomedical applications. However, engineered tissue scaffolds composed of PU have not found their way into clinical application, mainly due to the difficulty of balancing the control of material properties with the desired cellular response. A simple method for the synthesis of tunable bioactive poly(ethylene glycol) diacrylate (PEGDA) hydrogels containing photocurable PU is described. These hydrogels may be modified with PEGylated peptides or proteins to impart variable biological functions, and the mechanical properties of the hydrogels can be tuned based on the ratios of PU and PEGDA. Studies with human cells revealed that PU-PEG blended hydrogels support cell adhesion and viability when cell adhesion peptides are crosslinked within the hydrogel matrix. These hydrogels represent a unique and highly tailorable system for synthesizing PU-based synthetic extracellular matrices for tissue engineering applications.

Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates.

Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(epsilon-caprolactone-co-DL-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Young's modulus ranged from 1.20-1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.

Balancing the rates of new bone formation and polymer degradation enhances healing of weight-bearing allograft/polyurethane composites in rabbit femoral defects.

There is a compelling clinical need for bone grafts with initial bone-like mechanical properties that actively remodel for repair of weight-bearing bone defects, such as fractures of the tibial plateau and vertebrae. However, there is a paucity of studies investigating remodeling of weight-bearing bone grafts in preclinical models, and consequently there is limited understanding of the mechanisms by which these grafts remodel in vivo. In this study, we investigated the effects of the rates of new bone formation, matrix resorption, and polymer degradation on healing of settable weight-bearing polyurethane/allograft composites in a rabbit femoral condyle defect model. The grafts induced progressive healing in vivo, as evidenced by an increase in new bone formation, as well as a decrease in residual allograft and polymer from 6 to 12 weeks. However, the mismatch between the rates of autocatalytic polymer degradation and zero-order (independent of time) new bone formation resulted in incomplete healing in the interior of the composite. Augmentation of the grafts with recombinant human bone morphogenetic protein-2 not only increased the rate of new bone formation, but also altered the degradation mechanism of the polymer to approximate a zero-order process. The consequent matching of the rates of new bone formation and polymer degradation resulted in more extensive healing at later time points in all regions of the graft. These observations underscore the importance of balancing the rates of new bone formation and degradation to promote healing of settable weight-bearing bone grafts that maintain bone-like strength, while actively remodeling.

Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise among limits of detection, reduced time, and resources.

Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.

Synthesis, characterization of calcium phosphates/polyurethane composites for weight-bearing implants.

Calcium phosphate (CaP)/polymer composites have been studied as an alternative graft material for the treatment of bone defects. In this study, lysine-triisocyanate-based polyurethane (PUR) composites were synthesized from both hydroxyapatite (HA) and ß-tricalcium phosphate (TCP) to reduce the brittleness of CaP and increase the bioactivity of the polymer. The mechanical properties and in vitro cellular response were investigated for both HA/PUR and TCP/PUR composites. The composites were implanted in femoral defects in rats, and in vivo bioactivity was evaluated by X-rays, micro-computed tomography (µCT), and histological sections. In biomechanical testing, PUR improved the mechanical properties of the CaP, thus rendering it potentially suitable for weight-bearing applications. In vitro cell culture studies showed that CaP/PUR composites are biocompatible, with ß-TCP enhancing the cell viability and proliferation relative to HA. CaP/PUR composites also supported the differentiation of osteoblastic cells on the materials. When implanted in rat femoral defects, the CaP/PUR composites were biocompatible and osteoconductive with no adverse inflammatory response, as evidenced by X-rays, µCT images, and histological sections. Additionally, a histological examination showed evidence of cellular infiltration and appositional remodeling. These results suggest that CaP/PUR composites could be potentially useful biomaterials for weight-bearing orthopaedic implants.

Injectable reactive biocomposites for bone healing in critical-size rabbit calvarial defects.

Craniofacial injuries can result from trauma, tumor ablation, or infection and may require multiple surgical revisions. To address the challenges associated with treating craniofacial bone defects, an ideal material should have the ability to fit complex defects (i.e. be conformable), provide temporary protection to the brain until the bone heals, and enhance tissue regeneration with the delivery of biologics. In this study, we evaluated the ability of injectable lysine-derived polyurethane (PUR)/allograft biocomposites to promote bone healing in critical-size rabbit calvarial defects. The biocomposites exhibited favorable injectability, characterized by a low yield stress to initiate flow of the material and a high initial viscosity to minimize the adverse phenomena of extravasation and filter pressing. After injection, the materials cured within 10-12 min to form a tough, elastomeric solid that maintained mechanical integrity during the healing process. When injected into a critical-size calvarial defect in rabbits, the biocomposites supported ingrowth of new bone. The addition of 80 µg mL(-1) recombinant human bone morphogenetic protein-2 (rhBMP-2) enhanced new bone formation in the interior of the defect, as well as bridging of the defect with new bone. These observations suggest that injectable reactive PUR/allograft biocomposites are a promising approach for healing calvarial defects by providing both mechanical stability as well as local delivery of rhBMP-2.

Biocompatibility and chemical reaction kinetics of injectable, settable polyurethane/allograft bone biocomposites.

Injectable and settable bone grafts offer significant advantages over pre-formed implants due to their ability to be administered using minimally invasive techniques and to conform to the shape of the defect. However, injectable biomaterials present biocompatibility challenges due to the potential toxicity and ultimate fate of reactive components that are not incorporated in the final cured product. In this study the effects of stoichiometry and triethylenediamine (TEDA) catalyst concentration on the reactivity, injectability, and biocompatibility of two component lysine-derived polyurethane (PUR) biocomposites were investigated. Rate constants were measured for the reactions of water (a blowing agent resulting in the generation of pores), polyester triol, dipropylene glycol (DPG), and allograft bone particles with the isocyanate-terminated prepolymer using an in situ attenuated total reflection Fourier transform infrared spectroscopy technique. Based on the measured rate constants, a kinetic model predicting the conversion of each component with time was developed. Despite the fact that TEDA is a well-known urethane gelling catalyst, it was found to preferentially catalyze the blowing reaction with water relative to the gelling reactions by a ratio >17:1. Thus the kinetic model predicted that the prepolymer and water proceeded to full conversion, while the conversions of polyester triol and DPG were <70% after 24h, which was consistent with leaching experiments showing that only non-cytotoxic polyester triol and DPG were released from the reactive PUR at early time points. The PUR biocomposite supported cellular infiltration and remodeling in femoral condyle defects in rabbits at 8weeks, and there was no evidence of an adverse inflammatory response induced by unreacted components from the biocomposite or degradation products from the cured polymer. Taken together, these data underscore the utility of the kinetic model in predicting the biocompatibility of reactive biomaterials.

Synthesis and characterization of an injectable allograft bone/polymer composite bone void filler with tunable mechanical properties.

In recent years, considerable effort has been expended toward the development of synthetic bone graft materials. Injectable biomaterials offer several advantages relative to implants due to their ability to cure in situ, thus conforming to irregularly shaped defects. While Food and Drug Administration-approved injectable calcium phosphate cements have excellent osteoconductivity and compressive strengths, these materials have small pore sizes (e.g., 1 mum) and are thus relatively impermeable to cellular infiltration. To overcome this limitation, we aimed to develop injectable allograft bone/polyurethane (PUR) composite bone void fillers with tunable properties that support rapid cellular infiltration and remodeling. The materials comprised particulated (e.g., >100 microm) allograft bone particles and a biodegradable two-component PUR, and had variable (e.g., 30%-70%) porosities. The injectable void fillers exhibited an initial dynamic viscosity of 220 Pa.s at clinically relevant shear rates (40 s(-1)), wet compressive strengths ranging from < 1 to 13 MPa, working times from 3 to 8 min, and setting times from 10 to 20 min, which are comparable to the properties of calcium phosphate bone cements. When injected in femoral plug defects in athymic rats, the composites supported extensive cellular infiltration, allograft resorption, collagen deposition, and new bone formation at 3 weeks. The combination of both initial mechanical properties suitable for weight-bearing applications as well as the ability of the materials to undergo rapid cellular infiltration and remodeling may present potentially compelling opportunities for injectable allograft/PUR composites as biomedical devices for bone regeneration.

Synthesis, characterization, and remodeling of weight-bearing allograft bone/polyurethane composites in the rabbit.

The process of bone healing requires the restoration of both anatomy and physiology, and there is a recognized need for innovative biomaterials that facilitate remodeling throughout this complex process. While porous scaffolds with a high degree of interconnectivity are known to accelerate cellular infiltration and new bone formation, the presence of pores significantly diminishes the initial mechanical properties of the materials, rendering them largely unsuitable for load-bearing applications. In this study, a family of non-porous composites has been fabricated by reactive compression molding of mineralized allograft bone particles (MBPs) with a biodegradable polyurethane (PUR) binder, which is synthesized from a polyester polyol and a lysine-derived polyisocyanate. At volume fractions exceeding the random close-packing limit, the particulated allograft component presented a nearly continuous osteoconductive pathway for cells into the interior of the implant. By varying the molecular weight of the polyol and manipulating the surface chemistry of the MBP via surface demineralization, compressive modulus and strength values of 3-6 GPa and 107-172 MPa were achieved, respectively. When implanted in bilateral femoral condyle plug defects in New Zealand White rabbits, MBP/PUR composites exhibited resorption of the allograft and polymer components, extensive cellular infiltration deep into the interior of the implant, and new bone formation at 6 weeks. While later in vivo timepoints are necessary to determine the ultimate fate of the MBP/PUR composites, these observations suggest that allograft bone/polymer composites have potential for future development as weight-bearing devices for orthopedic applications.