Quantification of cell migration: metrics selection to model application.

Cell migration plays an essential role in physiological and pathological states, such as immune response, tissue generation and tumor development. This phenomenon can occur spontaneously or it can be triggered by an external stimuli, including biochemical, mechanical, or electrical cues that induce or direct cells to migrate. The migratory response to these cues is foundational to several fields including neuroscience, cancer and regenerative medicine. Various platforms are available to qualitatively and quantitatively measure cell migration, making the measurements of cell motility straight-forward. Migratory behavior must be analyzed by multiple metrics and then models to connect the measurements to physiological meaning. This review will focus on describing and quantifying cell movement for individual cell migration.

Anti-adhesive bioresorbable elastomer-coated composite hernia mesh that reduce intraperitoneal adhesions.

Intraperitoneal adhesions (IAs) are a major complication arising from abdominal repair surgeries, including hernia repair procedures. Herein, we fabricated a composite mesh device using a macroporous monofilament polypropylene mesh and a degradable elastomer coating designed to meet the requirements of this clinical application. The degradable elastomer was synthesized using an organo-base catalyzed thiol-yne addition polymerization that affords independent control of degradation rate and mechanical properties. The elastomeric coating was further enhanced by the covalent tethering of antifouling zwitterion molecules. Mechanical testing demonstrated the elastomer forms a robust coating on the polypropylene mesh does not exhibit micro-fractures, cracks or mechanical delamination under cyclic fatigue testing that exceeds peak abdominal loads (50 N/cm). Quartz crystal microbalance measurements showed the zwitterionic functionalized elastomer further reduced fibrinogen adsorption by 73% in vitro when compared to unfunctionalized elastomer controls. The elastomer exhibited degradation with limited tissue response in a 10-week murine subcutaneous implantation model. We also evaluated the composite mesh in an 84-day study in a rabbit cecal abrasion hernia adhesion model. The zwitterionic composite mesh significantly reduced the extent and tenacity of IAs by 94% and 90% respectively with respect to uncoated polypropylene mesh. The resulting composite mesh device is an excellent candidate to reduce complications related to abdominal repair through suppressed fouling and adhesion formation, reduced tissue inflammation, and appropriate degradation rate.

Degradable, Photochemically Printable Poly(propylene fumarate)-Based ABA Triblock Elastomers.

Additive manufacturing is rapidly advancing tissue engineering, but the scope of its clinical translation is limited by a lack of materials designed to meet specific mechanical properties and resorption timelines. Materials that are printable via photochemical cross-linking, fully degradable, and elastomeric have proven to be particularly challenging to develop. Herein, we report the synthesis of a series of poly(propylene fumarate-b-γ-methyl-ε-caprolactone-b-propylene fumarate) ABA triblock polymers using sequential ring-opening polymerization and ring-opening copolymerization. When cross-linked photochemically using a continuous liquid interface production digital light processing Carbon M2 printer, these ABA-type triblock copolymers are durable elastomers with tunable degradation and elastic properties. The polymers are shown to undergo slow, hydrolytic degradation in vitro with minimal loss of mechanical performance during degradation.

Concomitant control of mechanical properties and degradation in resorbable elastomer-like materials using stereochemistry and stoichiometry for soft tissue engineering.

Complex biological tissues are highly viscoelastic and dynamic. Efforts to repair or replace cartilage, tendon, muscle, and vasculature using materials that facilitate repair and regeneration have been ongoing for decades. However, materials that possess the mechanical, chemical, and resorption characteristics necessary to recapitulate these tissues have been difficult to mimic using synthetic resorbable biomaterials. Herein, we report a series of resorbable elastomer-like materials that are compositionally identical and possess varying ratios of cis:trans double bonds in the backbone. These features afford concomitant control over the mechanical and surface eroding degradation properties of these materials. We show the materials can be functionalized post-polymerization with bioactive species and enhance cell adhesion. Furthermore, an in vivo rat model demonstrates that degradation and resorption are dependent on succinate stoichiometry in the elastomers and the results show limited inflammation highlighting their potential for use in soft tissue regeneration and drug delivery.

Mechanically tunable, human mesenchymal stem cell viable poly(ethylene glycol)-oxime hydrogels with invariant precursor composition, concentration, and stoichiometry.

Hydrogels are used widely for exploratory tissue engineering studies. However, currently no hydrogel systems have been reported that exhibit a wide range of elastic modulus without changing precursor concentration, identity, or stoichiometry. Herein, ester and amide-based PEG-oxime hydrogels with tunable moduli (~5-30 kPa) were synthesized with identical precursor mass fraction, stoichiometry, and concentration by varying the pH and buffer concentration of the gelation solution, exploiting the kinetics of oxime bond formation. The observed modulus range can be attributed to increasing amounts of network defects in slower forming gels, as confirmed by equilibrium swelling and small angle neutron scattering (SANS) experiments. Finally, hMSC viability was confirmed in these materials in a 24 h assay. While only an initial demonstration of the potential utility, the controlled variation in defect density and modulus is an important step forward in isolating system variables for hypothesis-driven biological investigations.

Modulating Bioglass Concentration in 3D Printed Poly(propylene fumarate) Scaffolds for Post-Printing Functionalization with Bioactive Functional Groups.

Poly(propylene fumarate) (PPF) has shown potential for the treatment of bone defects as it can be 3D printed into scaffolds to suit patient-specific needs with strength comparable to that of bone. However, the lack of specific cell attachment and osteogenic signaling moieties have limited their utility as it is necessary to provide these signals to aid in bone tissue formation. To address this issue and provide a platform for functionalization, Bioglass (∼1-2 µm) microparticles have been incorporated into PPF to create a 3D printable resin with concentrations ranging from 0 to 10 wt %. The zero-shear viscosity of PPF-Bioglass resins increased proportionally from 0 to 2.5 wt % Bioglass, with values of 0.22 and 0.34 Pa·s, respectively. At higher Bioglass concentrations, 5 and 10 wt %, the resin viscosity increased to 0.44 and 1.31 Pa·s, exhibiting a 2- and 6-fold increase from the 0 wt % Bioglass resin. Despite this increase in viscosity, all resins remained printable with no print failures. In addition, the surface available Bioglass can tether catechol containing molecules for postprinting functionalization. Analysis of PPF-Bioglass functionalization using a catechol dye analyte shows functionalization increases with Bioglass concentration, up to 157 nmol/cm2, and demonstrates it is possible to modulate functionalization. This presents a versatile and highly translationally relevant strategy to functionalize 3D printed scaffolds post printing with a diverse array of functional species.

Zwitterionic amino acid-based Poly(ester urea)s suppress adhesion formation in a rat intra-abdominal cecal abrasion model.

Hernia repair outcomes have improved with more robust material options for surgeons and optimized surgical techniques. However, ventral hernia repairs remain challenging with an inherent risk of post-surgical adhesions in the peritoneal space which can occur regardless of interventional material or its surgical placement. Herein, amino acid-based poly(ester urea)s (PEUs) with varied amount of an allyl ether side chains were modified post polymerization modification with the zwitterionic sulfnate group (3-((3-((3-mercaptopropanoyl)oxy)propyl) dimethylammonio)propane-1-sulfonate) to promote anti-adhesive properties. These alloc-PEUs were processed using roll-to-roll fabrication methods to afford films that were amenable to surface functionalization via a zwitterion-thiol. Functional group availability on the surface was confirmed via fluorescence microscopy, x-ray photoelectron spectroscopy (XPS), and quartz crystal microbalance (QCM) measurements. Zwitterionic treated PEUs exhibited reduced fibrinogen adsorption in vitro when compared to unfunctionalized control polymer. A rat intrabdominal cecal abrasion adhesion model was used to assess the extent and tenacity of adhesion formation in the presence of the PEUs. The 10% alloc-PEU zwitterion functionalized material was found to reduce the extent and tenacity of adhesions when compared to adhesion controls and the unfunctionalized PEU controls.

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.

Optimization of photocrosslinkable resin components and 3D printing process parameters.

The role of 3D printing in the biomedical field is growing. In this context, photocrosslink-based 3D printing procedures for resorbable polymers stand out. Despite much work, more studies are needed on photocuring stereochemistry, new resin additives, new polymers and resin components. As part of these studies it is vital to present the logic used to optimize the amount of each resin constituent and how that effects printing process parameters. The present manuscript aims to analyze the effects of poly(propylene fumarate) (PPF) resin components and their effect on 3D printing process parameters. Diethyl fumarate (DEF), bisacylphosphine oxide (BAPO), Irgacure 784, 2-hydroxy-4-methoxybenzophenone (HMB) and, for the first time, in biomedical 3D printing, ethyl acetate (EA), were the resin components under investigation in this study. Regarding printing process parameters, Exposure Time, Voxel Depth, and Overcuring Depth were the parameters studied. Taguchi Design of Experiments was used to search for the effect of varying these resin constituent concentrations and 3D printing parameters on the curing behavior of 3D printable PPF resins. Our results indicate that resins with higher polymer cross-link density, especially those with a higher content of PPF, are able to be printed at higher voxel depth and with greater success (i.e., high yield). High voxel depth, as long as it does not sacrifice required resolution, is desirable as it speeds printing. Nevertheless, the overall process is governed by the correct setup of the voxel depth in relation to overcuring depth. In regards to resin biocompatibility, it was observed that EA is more effective than DEF, the material we had previously relied on. Our preliminary in vitro cytotoxicity tests indicate that the use of EA does not reduce scaffold biocompatibility as measured by standard cytotoxicity testing (i.e., ISO 10993-5). We demonstrate a workpath for resin constituent concentration optimization through thin film tests and photocrosslinkable process optimization. STATEMENT OF SIGNIFICANCE: We report here the results of a study of photo-crosslinkable polymer resin component optimization for the 3D printing of resorbable poly(propylene fumarate) (PPF) scaffolds. Resin additives are initially optimized for PPF thin film printing. Once those parameters have been optimized the 3D printing process parameters for PPF objects with complex, porous shapes can be optimized. The design of experiments to optimize both polymer thin films and complex porous resorbable polymer scaffolds is important as a guess and check, or in some cases a systematic method, are very likely to be too time consuming to accomplish. Previously unstudied resin components and process parameters are reported.

Enhancing Schwann cell migration using concentration gradients of laminin-derived peptides.

Neuroregeneration following peripheral nerve injury is largely mediated by Schwann cells (SC), the principal glial cell that supports neurons in the peripheral nervous system. Axonal regeneration in vivo is limited by the extent of SC migration into the gap between the proximal and distal nerve, however, little is known regarding the principal driving forces for SC migration. Engineered microenvironments, such as molecular and protein gradients, play a role in the migration of many cell types, including cancer cells and fibroblasts. However, haptotactic strategies have not been applied widely to SC. Herein, a series of tethered laminin-derived peptides were analyzed for their influence on SC adhesion, proliferation, and alignment. Concentration gradient substrates were fabricated using a controlled vapor deposition method, followed by covalent peptide attachment via a thiol-ene reaction, and characterized by X-ray photoelectron spectroscopy (XPS) and MALDI-MS imaging. While tethered RGD peptides supported SC adhesion and proliferation, concentration gradients of RGD had little influence on biased SC directional migration. In contrast, YIGSR promoted less SC attachment than RGD, yet YIGSR peptide gradients directed migration with a strong bias to the concentration profile. With YIGSR peptide, overall speed increased with the steepness of the peptide concentration profile. YIGSR gradients had no haptotactic effect on rat dermal fibroblast migration, in contrast to fibroblast migration on RGD gradients. The response of SC to these tethered peptide gradients will guide the development of translationally relevant constructs designed to facilitate endogenous SC infiltration into defects for nerve regeneration.

RGD-Modified Nanofibers Enhance Outcomes in Rats after Sciatic Nerve Injury.

Nerve injuries requiring surgery are a significant problem without good clinical alternatives to the autograft. Tissue engineering strategies are critically needed to provide an alternative. In this study, we utilized aligned nanofibers that were click-modified with the bioactive peptide RGD for rat sciatic nerve repair. Empty conduits or conduits filled with either non-functionalized aligned nanofibers or RGD-functionalized aligned nanofibers were used to repair a 13 mm gap in the rat sciatic nerve of animals for six weeks. The aligned nanofibers encouraged cell infiltration and nerve repair as shown by histological analysis. RGD-functionalized nanofibers reduced muscle atrophy. During the six weeks of recovery, the animals were subjected to motor and sensory tests. Sensory recovery was improved in the RGD-functionalized nanofiber group by week 4, while other groups needed six weeks to show improvement after injury. Thus, the use of functionalized nanofibers provides cues that aid in in vivo nerve repair and should be considered as a future repair strategy.

Poly(propylene fumarate)-based materials: Synthesis, functionalization, properties, device fabrication and biomedical applications.

Poly(propylene fumarate) (PPF) is a biodegradable polymer that has been investigated extensively over the last three decades. It has led many scientists to synthesize and fabricate a variety of PPF-based materials for biomedical applications due to its controllable mechanical properties, tunable degradation and biocompatibility. This review provides a comprehensive overview of the progress made in improving PPF synthesis, resin formulation, crosslinking, device fabrication and post polymerization modification. Further, we highlight the influence of these parameters on biodegradation, biocompatibility, and their use in a number of regenerative medicine applications, especially bone tissue engineering. In particular, the use of 3D printing techniques for the fabrication of PPF-based scaffolds is extensively reviewed. The recent invention of a ring-opening polymerization method affords precise control of PPF molecular mass, molecular mass distribution (ƉM) and viscosity. Low ƉM facilitates time-certain resorption of 3D printed structures. Novel post-polymerization and post-printing functionalization methods have accelerated the expansion of biomedical applications that utilize PPF-based materials. Finally, we shed light on evolving uses of PPF-based materials for orthopedics/bone tissue engineering and other biomedical applications, including its use as a hydrogel for bioprinting.

3D Printing of Poly(propylene fumarate) Oligomers: Evaluation of Resin Viscosity, Printing Characteristics and Mechanical Properties.

Complex three-dimensional (3D) pore geometries, useful for tissue engineering scaffolds, can be fabricated via photo-crosslinking of resorbable poly(propylene fumarate) (PPF) resins using stereolithography (SLA) and/or continuous digital light processing (cDLP) methods. Physico-chemical parameters inherent to 3D printable resin design, include viscosity, polymer concentration, degree of polymerization, and resin printing temperature. We report here on our study of these parameters and their influence the cDLP 3D printing process and the resulting mechanical properties. A series of PPF oligomers were synthesized by the ring-opening copolymerization (ROCOP) of maleic anhydride and propylene oxide followed by a base-catalyzed isomerization. The resin viscosities were measured as a function of number-average molecular mass ([Formula: see text]) of the PPF oligomers (1.1, 1.7 and 2.0 kDa), concentrations of PPF in the reactive diluent diethyl fumarate (DEF) (50 and 75 wt %) and resin temperature (25 to 55 °C). The zero-shear viscosity (η0) of the resins was found to be temperature-dependent and follow a linear Arrhenius relationship. Tensile tests demonstrated mechanical properties within the range of trabecular bone, with the ultimate strength at break above 15 MPa and elastic moduli between 178 and 199 MPa.

Amino acid-based Poly(ester urea) copolymer films for hernia-repair applications.

The use of degradable materials is required to address current performance and functionality shortcomings from biologically-derived tissues and non-resorbable synthetic materials used for hernia mesh repair applications. Herein a series of degradable l-valine-co-l-phenylalanine poly(ester urea) (PEU) copolymers were investigated for soft-tissue repair. Poly[(1-VAL-8)0.7-co-(1-PHE-6)0.3] showed the highest uniaxial mechanical properties (332.5 ±â€¯3.5 MPa). Additionally, l-valine-co-l-phenylalanine poly(ester urea)s were blade coated on small intestine submucosa extracellular matrix (SIS-ECM) and found to enhance the burst test mechanical properties of SIS-ECM in composite films (force at break between 102.6 ±â€¯6.5-151.4 ±â€¯11.3 N). Free standing films of l-valine-co-l-phenylalanine PEUs were found to have superior extension at break when compared to SIS-ECM (averages between 1.2 and 1.9 cm and 1.2 cm respectively). Fibroblast (L-929) spreading, proliferation, and improved attachment over control were observed without toxicity in vitro, while a reduced inflammatory response at both 7 and 14 days post-implant was observed for poly[(1-VAL-8)⁠0.7-co-(1-PHE-6)⁠0.3] when compared to polypropylene in an in vivo rat hernia model. These results support the use of PEU copolymers as free-standing films or as composite materials in soft-tissue applications for hernia-repair.

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.

pH-Responsive, Functionalizable Spyrocyclic Polycarbonate: A Versatile Platform for Biocompatible Nanoparticles.

Polymeric nanoparticles are widely investigated to enhance the selectivity of therapeutics to targeted sites, as well as to increase circulation lifetime and water solubility of poorly soluble drugs. In contrast to the encapsulation of the cargo into the nanostructures, the conjugation directly to the polymer backbone allows better control on the loading and selective triggered release. In this work we report a simple procedure to create biodegradable polycarbonate graft copolymer nanoparticles via a ring opening polymerization and subsequent postpolymerization modification strategies. The polymer, designed with both pH-responsive acetal linkages and a norbornene group, allows for highly efficient postpolymerization modifications through a range of chemistries to conjugate imaging agents and solubilizing arms to direct self-assembly. To demonstrate the potential of this approach, polycarbonate-based nanoparticles were tested for biocompatibility and their ability to be internalized in A549 and IMR-90 cell lines.

Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers.

Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. STATEMENT OF SIGNIFICANCE: In this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.

Enhanced Rotator-Cuff Repair Using Platelet-Rich Plasma Adsorbed on Branched Poly(ester urea)s.

Platelet-rich plasma (PRP) is a clinically relevant source of growth factors used commonly by surgeons. The clinical efficacy of PRP use as reported in the literature is widely variable which is likely attributed to poorly defined retention time of PRP at the repair site. To overcome this limitation, branched poly(ester urea) (PEU) nanofibers were used to adsorb and retain PRP at the implant site in an acute rotator-cuff tear model in rats. The adsorption of PRP to the branched-PEU 8% material was characterized using quartz crystal microbalance (QCM) and immuno-protein assay. After adsorption of PRP to the nanofiber sheet, the platelets actively released proteins. The adhesion of platelets to the nanofiber material was confirmed by immunofluorescence using a p-selectin antibody. In vivo testing using a rat rotator-cuff repair model compared five groups; no repair (control), suture repair only, repair with disc implant (Disc), repair with PRP-soaked disc (Disc PRP), and a PRP injection (PRP). Mechanical testing at 84 d for the four surgical repair groups resulted in a higher stiffness (11.8 ± 3.8 N/mm, 13.5 ± 3.8 N/mm, 16.8 ± 5.8 N/mm, 12.2 ± 2.6 N/mm, respectively) for the Disc PRP group. Histological staining using trichrome, hematoxylin, and eosin Y (H&E), and safranin O confirmed more collagen organization in the Disc PRP group at 21 and 84 d. Limited inflammation and recovery toward preoperative mechanical properties indicate PEU nanofiber discs as translationally relevant.

Preclinical in Vitro and in Vivo Assessment of Linear and Branched l-Valine-Based Poly(ester urea)s for Soft Tissue Applications.

New polymers are needed to address the shortcomings of commercially available materials for soft tissue repair. Herein, we investigated a series of l-valine-based poly(ester urea)s (PEUs) that vary in monomer composition and the extent of branching as candidate materials for soft tissue repair. The preimplantation Young's moduli (105 ± 30 to 269 ± 12 MPa) for all the PEUs are comparable to those of polypropylene (165 ± 5 MPa) materials currently employed in hernia-mesh repair. The 2% branched poly(1-VAL-8) maintained the highest Young's modulus following 3 months of in vivo implantation (78 ± 34 MPa) when compared to other PEU analogues (20 ± 6-45 ± 5 MPa). Neither the linear or branched PEUs elicited a significant inflammatory response in vivo as noted by less fibrous capsule formation after 3 months of implantation (80 ± 38 to 103 ± 33 µm) relative to polypropylene controls (126 ± 34 µm). Mechanical degradation in vivo over three months, coupled with limited inflammatory response, suggests that l-valine-based PEUs are translationally relevant materials for soft tissue applications.

Magnesium Catalyzed Polymerization of End Functionalized Poly(propylene maleate) and Poly(propylene fumarate) for 3D Printing of Bioactive Scaffolds.

The ring-opening copolymerization of maleic anhydride and propylene oxide, using a functionalized primary alcohol initiator and magnesium 2,6-di-tert-butyl phenoxide as a catalyst, was investigated in order to produce high end-group fidelity poly(propylene maleate). Subsequent isomerization of the material into 3D printable poly(propylene fumarate) was utilized to produce thin films and scaffolds possessing groups that can be modified with bioactive groups postpolymerization and postprinting. The surface concentration of these modifiable groups was determined to be 30.0 ± 3.3 pmol·cm-2, and copper-mediated azide-alkyne cycloaddition was used to attach a small molecule dye and cell adhesive GRGDS peptides to the surface as a model system. The films were then studied for cytotoxicity and found to have high cell viability before and after surface modification.