Research

Research Summary:
The Becker Laboratory for Functional Biomaterials has evolved over the last eight years into a multidisciplinary research team that is developing soft matter solutions for unmet medical needs. While working across a number of translational platforms, we are collectively focused on translationally relevant method development that affords the precise placement of bioactive species on degradable polymers. Our “retrosynthetic” approach to materials development begins with trying to understand the clinical presentation of a problem, understanding the biotic-abiotic interface at a molecular level and formulating approaches that involve presenting a functional synthetic construct with the appropriate physico-chemical and mechanical properties that addresses or mitigates the problem. While this description is a gross oversimplification of the hypothesis-driven materials development process we pursue, it highlights the fundamental, chemical approach that differentiates us from many others working in the biomaterial space.

We are focused in these key areas which motivate our work:

PHOTOCHEMICAL 3D PRINTING FOR REGENERATIVE MEDICINE

Additive manufacturing has the potential to change medicine but the diversity of degradable materials available for use in regenerative medicine application is limited. The production of customized geometrically complex scaffolds that are resorbable, support the required biomechanical loads and are functionalized with bioactive species will require highly specialized polymers. Each of the 3D printing techniques currently available is highly dependent on the chemical, thermal and viscosity properties of the polymers used as the feedstock. Continuous digital light processing (cDLP) is a stereolithographic method that affords fine features (<50 µm) using fluidic photo-cross-linkable polymer resins. However the number of resorbable resins that can be photochemically crosslinked is very limited. Poly(propylene fumarate) (PPF), is an unsaturated, photo-cross-linkable polyester. Upon hydrolysis of the ester linkage PPF degrades into two bioresorbable products, fumaric acid and propylene glycol, on a time scale consistent with the rate of bone growth. Since its discovery in 1994, PPF has been synthesized using a step-growth polycondensation of fumaric acid and propylene glycol and has had success in preclinical studies for controlled drug release, vascular stents, nerve grafts, and tissue engineering of bone and cartilage The condensation polymerization route requires high energy input, long reaction times, and results in low conversion (~35%), uncontrolled molecular mass distribution, conjugate-addition side reactions, and unwanted crosslinking.

In 2015, we discovered a route that improved upon the step-growth method by utilizing a ring-opening copolymerization (ROP) of maleic anhydride and propylene oxide to yield poly(propylene maleate) (PPM), which can be isomerized into PPF using a weak base. This method affords a high degree of control over molecular mass, molecular mass distribution, and end-group fidelity by utilizing magnesium 2,6-di¬-tert¬-butyl phenoxide (Mg(BHT)2¬(THF)2), a catalyst commonly used for the “immortal” ring opening polymerization of lactones.

Incorporation of other monomers and polymers into PPF resin systems is thought to be a viable method of tailoring materials to specific purposes. Among countless other examples of polyester copolymerization, ε-caprolactone (εCL) has been copolymerized with α-propargyl-ε-caprolactone as a method of introducing macromolecular contrast agents to enhance magnetic resonance visibility via a copper-mediated azide-alkyne cycloaddition (CuAAC) and ω-pentadecalactone (PDL) block-like copolymers have been synthesized with epsilon substituted lactones in order to make a functional, ‘green’ alternative to low-density polyethylene. We proposed to modify PPF properties by creating a polymer blend containing poly(caprolactone fumarate) to tune the thermal, mechanical, and rheological properties and by incorporating εCL into PPF via a three-step polycondensation reaction of oligomeric materials in order to modify crosslinking temperature, gelation time, and mechanical properties. In this effort, we are developing a sequential ring-opening polymerization (ROP) and ROCOP synthesis using (Mg(BHT)2¬(THF)2) to create one-pot PPF block-copolymers with cyclic polyesters for additive manufacturing and tissue engineering applications.

The foundational PCT application outlining the new PPF synthesis was recently licensed by 21MedTech to deliver first in man clinical trials for PPF-based scaffolds for mandibular defect repair next year. While this is a very recent development in the research efforts of my group, it is advancing rapidly toward commercialization and clinical impact. This work is funded by The Ohio Third Frontier and 21st Century Medical Technologies.

Relevant Publications
1) EP Childers, M Wang, JP Fisher, ML Becker, D Dean* “3D Printing of Resorbable Poly(propylene fumarate) Tissue Engineering Scaffolds” MRS Bulletin 2015, 40(02) 119-126.
2) Y Xu ‡, D Luong‡, K Martin, H Lara-Padilla, JM Walker, AP Kleinfehn, D Dean, ML Becker*, “Modification of Poly(Propylene Fumarate)-Bioglass® Composites with Peptide Conjugates to Enhance Bioactivity”, Biomacromolecules 2017, in press.
3) JM Walker, E Bodamer, AP Kleinfehn, Y Luo, ML Becker, D Dean “Design and mechanical characterization of solid and highly porous 3D printed poly(propylene fumarate) scaffolds”, Progress in Additive Manufacturing, 2017, 2, 99-108.
4) JM Walker, E Bodamer, O Krebs†, Y Lou, A Kleinfehn, ML Becker, D Dean “Effect of chemical and physical properties on the in vitro degradation of 3D printed high resolution poly(propylene fumarate) scaffolds” Biomacromolecules, 2017, 18(4): 1419-1425.
5) JA Wilson, S Peterson, Y Chen, ML Becker* “Well defined block copolymers of polylactones and poly(propylene fumarate) 2017, submitted.
6) JA Wilson, Y Chen, ML Becker* “Copolymerization of functional epoxides with maleic anhydride 2017, submitted.
7) JA Wilson, AP Kleinfehn, D Luong, S Sallam, C Wesdemiotis, ML Becker* “Magnesium catalyzed polymerization of end functionalized poly(propylene maleate) and poly(propylene fumarate) for 3D Printing” 2017, submitted.

AMINO ACID BASED POLY(ESTER UREA)S FOR REGENERATIVE MEDICINE

The development of a fundamental understanding of the role of architecture and the influence of stoicheometry and chirality on the physical and chemical properties is critical to the development of new degradable materials. There are a number of biodegradable polymers including poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(glycolide) (PGA), and copolymers thereof that used commercially and while their properties in vitro and in vivo are largely understood, their range of physical and chemical properties is somewhat limited. There are number of other, albeit less well known, degradable polymers used clinically including polyurethanes (PU), poly(orthoesters), poly(carbonates), poly(ester urea) (PEU), and more recently poly(-amino acids). An increasingly common strategy has been to use naturally occurring amino acids as building blocks for monomer precursors.

While these classes of materials have had a transformative influence on the design of next generation materials for regenerative medicine, in general, the mechanical properties have been insufficient for load bearing applications. The maximum reported mechanical properties of non-filled degradable polymers varies between 0.9-2.5 GPa (Young’s Modulus) depending on the test method and thermal history. For comparison, the elastic modulus of cortical bone within the mid-diaphysis of a long bone is approximately 18 GPa. Another significant limitation is that semi-crystalline polyesters, PCL and PLLA, degrade very slowly. It can take years for these materials to degrade and resorb fully and the associated acidification that results often leads to inflammation. Degradable polymers that possess high moduli and relative larger degradation rates are needed for numerous regenerative medicine and orthopedic applications. α-Amino acid-based poly(ester urea)s have proven to be important materials for biomedical applications because of their excellent blood, tissue compatibility and non-toxic hydrolysis byproducts. Also, their semi-crystalline structure provides a non-chemical method to enhance their mechanical properties and processing characteristics.

Amino acid-based poly(ester urea)s are a diverse library of materials which uniquely do not lead to acidosis upon degradation. This distinct divergence from other polyester materials is likely due to the presence of the conjugate base (urea) present in each repeat unit that serve to neutralize the degrading ester linkages. The mechanical properties are three-fold higher than the highest reported values for lactic acid. My team has developed a number of non-canonical amino acid synthetic strategies, architectures and functionalization methods that further enhance the bioactivity, degradation and mechanical properties. We have extensively explored the chemical and mechanical properties of this class of material in addition to more recent work involving defining the shape memory properties using the unique hydrogen bonding properties of these materials.

In recent publications we have described the synthesis and characterization of a series of linear amino acid-based poly(ester urea)s that are strong and biodegradable. We have systematically varied the diol chain length and measured the mechanical properties and in vitro biodegradation rate of the polymers. We have found a wide ranging variation in physical and chemical properties depending on the nature of the amino acid. Some of the variations are subtle while others within simple changes in diol length can alter the Young’s modulus by as much as two orders of magnitude. We have also shown that a 1,6-hexandiol L-phenylalanine-based poly(ester urea) (poly(1-PHE-6)) possesses mechanical properties that greatly exceed commonly utilized degradable polyesters. Poly(1-PHE-6) was found to have a storage modulus of 6.8 GPa in our study, which is nearly twice the reported value of poly(lactic acid).

There has been no evidence of inflammation due to implant acidification when poly(1-PHE-6) was implanted in vivo. This trend has held up in a number of animal models including rats, rabbits and sheep. To note the utility of our materials in applications of particular relevance to the Army, a phenylalanine-based PEU material synthesized by our laboratory was used by The Methodist Hospital Research Institute in Houston as a part of a DARPA subcontract. The materials were used to repair a segmental bone defect repair in a sheep model that serves as a surrogate clinical model for injuries to warfighters due to high velocity projective injuries and improvised explosive devices. We have also initiated translational efforts in burns and blood vessel regeneration and are pursuing funding for these efforts at the appropriate agencies.

In the last year, we have completed in vivo studies in rats (vascular grafts and bone-tendon interface healing), rabbits (protein delivery from nanofibers) and sheep (segmental bone defect) and the results of these studies just being published in the literature.

These results will be the foundation of our translational research efforts over the next five years. This work is supported by the National Science Foundation, the Department of Defense, Merck and Cook Medical. The largest award to date ($6M) from the Department of Defense is a distinctive partnership with the Houston Methodist Research Institute and is designed to optimize our designs in a large sheep trial and includes a pilot exemption for use in military soldiers who present with a significant injury.

Relevant Publications
1) S Li, J Yu, MB Wade, GM Policastro, ML Becker* “Radiopaque, Iodine Functionalized Poly(ester urea)s” Biomacromolecules 2015, 16(2), 615–624.
2) GM Policastro, F Lin, LA Smith Callahan, A Esterle, M Graham, KS Stakleff, ML Becker* “OGP Functionalized Phenylalanine-based Poly(ester urea) for Enhancing Osteoinductive Potential of human Mesenchymal Stem Cells” Biomacromolecules 2015, 16(4), 1358-1371.
3) V Bhagat, ML Becker* “Degradable Adhesives for Surgery and Tissue Engineering” Biomacromolecules, 2017, in press.
4) GI Peterson, AV Dobrynin, ML Becker* Shape Memory Materials in Medicine, Advanced Healthcare Materials, 2017, in press.
5) Y Gao, Y Xu, A Land, JT Harris†, GM Policastro, T Ritzman*, J Bundy, ML Becker*, “Extended delivery of hGH from polymeric nanofibers” ACS Macro Lett., 2017, 6, 875–880.
6) S Li, Y Xu, J Yu, ML Becker* “Enhanced Osteogenic Activity of Poly(ester urea) Scaffolds using Facile Post-3D Printing Peptide Functionalization Strategies”, Biomaterials 2017, 141, 176-187.

POST-ELECTROSPINNING FUNCTIONALIZATION OF POLYMER NANOFIBERS

The Becker group has made seminal advances on the development of translationally-relevant methods for attaching bioactive groups to degradable polymer nanofibers. Many groups have worked on creating peptide functional materials for regenerative medicine. Similarly, electrospun nanofibers have emerged over the past three decades as valuable tools for high surface area applications in nerve repair as well as wound healing and vascular tissue engineering applications. However, it is well known that attaching the bioactive groups prior to fabrication results in a significant fraction of the bioactive species buried (and not bioavailable) within the bulk of the fiber. As most cellular interactions are surface dominated, presentation of the optimal concentration of bioactive groups is critical to advancing these materials to the stage where they are clinically useful. The Becker Lab has shown the ability to precisely control the placement of three functional species on nanofiber substrates.

Mimicking the architecture of tissue and extracellular matrix (ECM) is one of the major hurdles to advancing regenerative medicine clinically. The development of polymeric nanofibers has greatly enhanced the potential for fabricating scaffolds that can potentially meet this challenge. Polymer nanofibers are fabricated using melt- or electrospinning processes that are able to control both size and morphology using a variety of conditions including solvent, concentration, and additives. Nanofibers, irrespective of their method of synthesis, have provided for scaffolds with high surface area and enhanced porosity. In both in vitro and in vivo clinical applications, these properties have shown a significant effect on cell adhesion, proliferation, and differentiation. Hence nanofibrous matrices are currently being explored as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, neural tissue engineering, vascular tissue engineering, and controlled delivery of drugs, proteins, and DNA.
The results of all these studies clearly indicate that nanofiber-based scaffolds show potential for a variety of tissue engineering applications. However, lack of scalable methods for derivatizing these fibers with peptides or proteins that interface with biological host at the cellular level have limited industrial adoption of the technology. Many of the reported nanofiber technologies have focused on using materials utilized in existing FDA-approved applications. However, synthetic polymers lack specific functionality which would guide or direct specific biological functions. The derivatization of nanofibers often requires multiple procedures each of which are time and resource intensive to optimize and may lead to immune specific reactions and biocompatibility problems.

Recently we have developed strain-promoted azide-alkyne cycloaddition (SPAAC) reactions that enable the use of dibenzocyclooctynol (DIBO) as a polymerization initiator for ring opening polymerizations. DIBO has been successfully used for the synthesis of degradable polylactides, poly(e-caprolactone) and mixed keto- polyesters.

In more recent work we have been developing novel nanofiber based substrates for embryonic stem cell expansion. Substrates for embryonic stem cell culture are typified by xenogenic, whole proteins or cells that are difficult and expensive to generate, characterize, and recapitulate. We have generated well-controlled, synthetic scaffolds of DIBO-functionalized poly(ԑ-caprolactone) (PCL) aligned nanofibers modified post-electrospinning with Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide via strain promoted alkyne-azide cycloaddition to control neural differentiation of mESC. GYIGSR-modified aligned nanofibers were used as substrates for 14 day neural differentiation of D3 mouse embryonic stem cells (mESC). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation profile than cells on laminin. GYIGSR-functionalized nanofiber substrates produced an increased gene expression of SOX1, a neural progenitor cell marker, and MAP2, CDH2, SYP, neuronal cell markers, at early time points. In addition, guidance of neurites was found along the fiber direction. Therefore, this work demonstrates the synthesis of novel synthetic polymer, that can be formed into a well-controlled, xeno-free synthetic nanofiber scaffold to replace currently used xenogenic and complex matrixes for neural differentiation of stem cells. This work is supported by the National Science Foundation and the National Institutes of Health.

Relevant Publications
1) J Zheng, K Liu, DH Reneker, ML Becker “Post-Assembly Derivatization of Electrospun Nanofibers via Strain-Promoted Azide Alkyne Cycloaddition” J. Amer. Chem. Soc., 2012, 134(41), 17274-17277.
2) LA Smith Callahan, S Xie, I Barker, J Zheng, DH Reneker, AP Dove, ML Becker “Accelerated Differentiation and Neurite Extension of mESC on Aligned Poly(lactide) Nanofibers Functionalized with YIGSR” Biomaterials, 2013, 34, 9089-9095.
3) J Zheng, G Hua, F Lin, J Yu, MB Wade, DH Reneker, ML Becker* “Post-electrospinning “Tri-Click” Functionalization of Degradable Polymer Nanofibers” ACS Macro Letters 2015, 4, 207-213.

PROGRAMMABLE FUNCTIONAL HYDROGELS

Human mesenchymal stem cells (hMSCs) have been demonstrated in multiple studies to respond to the rigidity of their microenvironment through changes in their genomic signaling, and consequently undergoing proliferation and differentiation events. As a result, there has been significant effort in controllably altering the mechanical properties of synthetic scaffolds to direct hMSC behavior. One particular area of focus has been in tunable hydrogel systems that mimic the mechanical features of selected tissues. For example, Discher et al. demonstrated control of hMSC differentiation based on the elastic modulus of a hydrogel scaffold. However, these and other systems generally require chemical or structural modifications in order to tune the moduli, methods for which have included altering precursor concentration or stoichiometry to modulate the effective cross-link density, incorporating an additional or modified cross-linking agent, changing precursor chain lengths to vary the molecular weight between cross-links, and changing the hydrogel composition altogether. These changes create challenges in the interpretation and control of cell studies. This project seeks to overcome these limitations by utilizing a kinetically-dependent cross-linking reaction that produces mechanically distinct hydrogels while simultaneously maintaining precursor composition, concentration, and stoichiometry. This work is supported by the National Institutes of Health.

Relevant Publications
1) Z Zander, G Hua, C Weiner, BD Vogt, ML Becker* “Control of Mesh Size and Modulus by Kinetically Dependent Cross‐Linking in Hydrogels” Advanced Materials, 2015, 27 (40), 6283-6288.
2) F Lin, J Yu, W Tang, J Zheng, A Defante, K Guo, C Wesdemiotis, ML Becker* “Peptide Functionalized Oxime Hydrogels with Tunable Mechanical Properties and Gelation Behavior” Biomacromolecules 2013, 34, 9089-9095.
3) J Zheng, LA Smith Callahan, J Hao, K Guo, C Wesdemiotis, RA Weiss, ML Becker* “Strain-Promoted Crosslinking of PEG-based Hydrogels via Copper-Free Cycloaddition” ACS Macro Letters, 2012, 1(8), 1071-1073.

PEPTIDE CONCENTRATION GRADIENTS FOR ASSESSING BIOAVAILABILITY

The biomaterials community also clearly understands that the molecular presentation and spatial distribution of peptides and growth factors can dramatically influence many important aspects of cell behavior and threshold concentrations are often necessary to trigger certain cellular responses. However, most synthetic tissue engineering constructs involve only one peptide or growth factor. In the last decade, my group has pioneered the development of gradient fabrication and characterization strategies that enable the concentration profiling of bioactive molecules on surfaces. The concentration ranges cover most of the useful ranges that are important in integrin mediated interactions. We recently demonstrated the synergistic acceleration of hMSC proliferation and osteoblast differentiation on 2D substrates possessing orthogonal concentration gradient of RGD and BMP-2 peptides was shown to be concentration dependent. The results, obtained without the use of osteoinductive additives, illustrated the concentration dependence and spatio-temporal features of the tethered peptides effects over the hMSC behavior that has not been demonstrated previously. This work is supported by the National Institutes of Health.

Relevant Publications
1) NM Moore, NJ Lin, ND Gallant, ML Becker* “The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation” Biomaterials, 2010, 31(7), 1604-1611.
2) NM Moore, NJ Lin, ND Gallant, ML Becker* “Synergistic Enhancement of Human Bone Marrow Stromal Cell Proliferation and Osteogenic differentiation on BMP-2 and RGD Peptide Concentration Gradients.” Acta Biomaterialia, 2011, 7, 2019-2100.
3) Y Ma, J Zheng, EF Amond†, CM Stafford, ML Becker* “Facile Fabrication of “Dual Click” One- and Two-Dimensional Orthogonal Peptide Concentration Gradients” Biomacromolecules, 2013, 14, 665-671.
4) Y Ma^, GM Policastro^, Q Li, J Zheng, R Jaquet, WJ Landis, ML Becker* “Concentration Dependent Differentiation of hMSC on orthogonal gradients of BMP-2 and GRGDS peptides” Biomacromolecules, 2016, 17, 1486−1495. (^co-first authors)