Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Poly(Thioketal Urethane) Autograft Extenders in an Intertransverse Process Model of Bone Formation.

IMPACT STATEMENT: The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.

Settable polymer/ceramic composite bone grafts stabilize weight-bearing tibial plateau slot defects and integrate with host bone in an ovine model.

Bone fractures at weight-bearing sites are challenging to treat due to the difficulty in maintaining articular congruency. An ideal biomaterial for fracture repair near articulating joints sets rapidly after implantation, stabilizes the fracture with minimal rigid implants, stimulates new bone formation, and remodels at a rate that maintains osseous integrity. Consequently, the design of biomaterials that mechanically stabilize fractures while remodeling to form new bone is an unmet challenge in bone tissue engineering. In this study, we investigated remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep. Nanocrystalline hydroxyapatite-poly(ester urethane) (nHA-PEUR) hybrid polymers were augmented with either ceramic granules (85% ß-tricalcium phosphate/15% hydroxyapatite, CG) or a blend of CG and bioactive glass (BG) particles to form a settable bone cement. The initial compressive strength and fatigue properties of the cements were comparable to those of non-resorbable poly(methyl methacrylate) bone cement. In animals that tolerated the initial few weeks of early weight-bearing, CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 weeks. In contrast, cements incorporating BG particles resorbed with fibrous tissue filling the defect. Furthermore, CG/nHA-PEUR cements remodeled significantly faster at the full weight-bearing tibial plateau site compared to the mechanically protected femoral condyle site in the same animal. These findings are the first to report a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near an articulating joint.

Injectable and compression-resistant low-viscosity polymer/ceramic composite carriers for rhBMP-2 in a rabbit model of posterolateral fusion: a pilot study.

BACKGROUND: The challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model. METHODS: LV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% ß-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts' capability to encourage spinal fusion. RESULTS: These materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 µg rhBMP-2/ml. Images of µCT and histological sections revealed evidence of bone fusion near the transverse processes. CONCLUSION: This study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion.

Remodeling of injectable, low-viscosity polymer/ceramic bone grafts in a sheep femoral defect model.

Ceramic/polymer composite bone grafts offer the potential advantage of combining the osteoconductivity of ceramic component with the ductility of polymeric component, resulting in a graft that meets many of the desired properties for bone void fillers (BVF). However, the relative contributions of the polymer and ceramic components to bone healing are not well understood. In this study, we compared remodeling of low-viscosity (LV) ceramic/poly(ester urethane) composites to a ceramic BVF control in a sheep femoral condyle plug defect model. LV composites incorporating either ceramic (LV/CM) or allograft bone (LV/A) particles were evaluated. We hypothesized that LV/CM composites which have the advantageous handling properties of injectability, flowability, and settability would heal comparably to the CM control, which was evaluated for up to 2 years to study its long-term degradation properties. Remodeling of LV/CM was comparable to that observed for the CM control, as evidenced by new bone formation on the surface of the ceramic particles. At early time points (4 months), LV/CM composites healed similar to the ceramic clinical control, while LV/A components showed more variable healing due to osteoclast-mediated resorption of the allograft particles. At longer time points (12-15 months), healing of LV/CM composites was more variable due to the nonhomogeneous distribution and lower concentration of the ceramic particles compared to the ceramic clinical control. Resorption of the ceramic particles was almost complete at 2 years. This study highlights the importance of optimizing the loading and distribution of ceramic particles in polymer/ceramic composites to maximize bone healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2333-2343, 2017.

Oxidatively Degradable Poly(thioketal urethane)/Ceramic Composite Bone Cements with Bone-Like Strength.

Synthetic bone cements are commonly used in orthopaedic procedures to aid in bone regeneration following trauma or disease. Polymeric cements like PMMA provide the mechanical strength necessary for orthopaedic applications, but they are not resorbable and do not integrate with host bone. Ceramic cements have a chemical composition similar to that of bone, but their brittle mechanical properties limit their use in weight-bearing applications. In this study, we designed oxidatively degradable, polymeric bone cements with mechanical properties suitable for bone tissue engineering applications. We synthesized a novel thioketal (TK) diol, which was crosslinked with a lysine triisocyanate (LTI) prepolymer to create hydrolytically stable poly(thioketal urethane)s (PTKUR) that degrade in the oxidative environment associated with bone defects. PTKUR films were hydrolytically stable for up to 6 months, but degraded rapidly (<1 week) under simulated oxidative conditions in vitro. When combined with ceramic micro- or nanoparticles, PTKUR cements exhibited working times comparable to calcium phosphate cements and strengths exceeding those of trabecular bone. PTKUR/ceramic composite cements supported appositional bone growth and integrated with host bone near the bone-cement interface at 6 and 12 weeks post-implantation in rabbit femoral condyle plug defects. Histological evidence of osteoclast-mediated resorption of the cements was observed at 6 and 12 weeks. These findings demonstrate that a PTKUR bone cement with bone-like strength can be selectively resorbed by cells involved in bone remodeling, and thus represent an important initial step toward the development of resorbable bone cements for weight-bearing applications.

Effects of Recombinant Human Bone Morphogenetic Protein-2 Dose and Ceramic Composition on New Bone Formation and Space Maintenance in a Canine Mandibular Ridge Saddle Defect Model.

Treatment of mandibular osseous defects is a significant clinical challenge. Maintenance of the height and width of the mandibular ridge is essential for placement of dental implants and restoration of normal dentition. While guided bone regeneration using protective membranes is an effective strategy for maintaining the anatomic contour of the ridge and promoting new bone formation, complications have been reported, including wound failure, seroma, and graft exposure leading to infection. In this study, we investigated injectable low-viscosity (LV) polyurethane/ceramic composites augmented with 100 µg/mL (low) or 400 µg/mL (high) recombinant human bone morphogenetic protein-2 (rhBMP-2) as space-maintaining bone grafts in a canine mandibular ridge saddle defect model. LV grafts were injected as a reactive paste that set in 5-10 min to form a solid porous composite with bulk modulus exceeding 1 MPa. We hypothesized that compression-resistant LV grafts would enhance new bone formation and maintain the anatomic contour of the mandibular ridge without the use of protective membranes. At the rhBMP-2 dose recommended for the absorbable collagen sponge carrier in dogs (400 µg/mL), LV grafts maintained the width and height of the host mandibular ridge and supported new bone formation, while at suboptimal (100 µg/mL) doses, the anatomic contour of the ridge was not maintained. These findings indicate that compression-resistant bone grafts with bulk moduli exceeding 1 MPa and rhBMP-2 doses comparable to that recommended for the collagen sponge carrier support new bone formation and maintain ridge height and width in mandibular ridge defects without protective membranes.

D-amino acid inhibits biofilm but not new bone formation in an ovine model.

BACKGROUND: Infectious complications of musculoskeletal trauma are an important factor contributing to patient morbidity. Biofilm-dispersive bone grafts augmented with D-amino acids (D-AAs) prevent biofilm formation in vitro and in vivo, but the effects of D-AAs on osteocompatibility and new bone formation have not been investigated. QUESTIONS/PURPOSES: We asked: (1) Do D-AAs hinder osteoblast and osteoclast differentiation in vitro? (2) Does local delivery of D-AAs from low-viscosity bone grafts inhibit new bone formation in a large-animal model? METHODS: Methicillin-sensitive Staphylococcus aureus and methicillin-resistant S aureus clinical isolates, mouse bone marrow stromal cells, and osteoclast precursor cells were treated with an equal mass (1:1:1) mixture of D-Pro:D-Met:D-Phe. The effects of the D-AA dose on biofilm inhibition (n = 4), biofilm dispersion (n = 4), and bone marrow stromal cell proliferation (n = 3) were quantitatively measured by crystal violet staining. Osteoblast differentiation was quantitatively assessed by alkaline phosphatase staining, von Kossa staining, and quantitative reverse transcription for the osteogenic factors a1Col1 and Ocn (n = 3). Osteoclast differentiation was quantitatively measured by tartrate-resistant acid phosphatase staining (n = 3). Bone grafts augmented with 0 or 200 mmol/L D-AAs were injected in ovine femoral condyle defects in four sheep. New bone formation was evaluated by µCT and histology 4 months later. An a priori power analysis indicated that a sample size of four would detect a 7.5% difference of bone volume/total volume between groups assuming a mean and SD of 30% and 5%, respectively, with a power of 80% and an alpha level of 0.05 using a two-tailed t-test between the means of two independent samples. RESULTS: Bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation were inhibited at D-AAs concentrations of 27 mmol/L or greater in a dose-responsive manner in vitro (p < 0.05). In methicillin-sensitive and methicillin-resistant S aureus clinical isolates, D-AAs inhibited biofilm formation at concentrations of 13.5 mmol/L or greater in vitro (p < 0.05). Local delivery of D-AAs from low-viscosity grafts did not inhibit new bone formation in a large-animal model pilot study (0 mmol/L D-AAs: bone volume/total volume = 26.9% ± 4.1%; 200 mmol/L D-AAs: bone volume/total volume = 28.3% ± 15.4%; mean difference with 95% CI = -1.4; p = 0.13). CONCLUSIONS: D-AAs inhibit biofilm formation, bone marrow stromal cell proliferation, osteoblast differentiation, and osteoclast differentiation in vitro in a dose-responsive manner. Local delivery of D-AAs from bone grafts did not inhibit new bone formation in vivo at clinically relevant doses. CLINICAL RELEVANCE: Local delivery of D-AAs is an effective antibiofilm strategy that does not appear to inhibit bone repair. Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects as a function of D-AA dose are required to further support the use of D-AAs in the clinical management of infected open fractures.

Effects of particle size and porosity on in vivo remodeling of settable allograft bone/polymer composites.

Established clinical approaches to treat bone voids include the implantation of autograft or allograft bone, ceramics, and other bone void fillers (BVFs). Composites prepared from lysine-derived polyurethanes and allograft bone can be injected as a reactive liquid and set to yield BVFs with mechanical strength comparable to trabecular bone. In this study, we investigated the effects of porosity, allograft particle size, and matrix mineralization on remodeling of injectable and settable allograft/polymer composites in a rabbit femoral condyle plug defect model. Both low viscosity and high viscosity grafts incorporating small (<105 µm) particles only partially healed at 12 weeks, and the addition of 10% demineralized bone matrix did not enhance healing. In contrast, composite grafts with large (105-500 µm) allograft particles healed at 12 weeks postimplantation, as evidenced by radial µCT and histomorphometric analysis. This study highlights particle size and surface connectivity as influential parameters regulating the remodeling of composite bone scaffolds.

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.

Investigating the Effects of Surface-Initiated Polymerization of ε-Caprolactone to Bioactive Glass Particles on the Mechanical Properties of Settable Polymer/Ceramic Composites.

Injectable bone grafts with strength exceeding that of trabecular bone could improve the management of a number of orthopaedic conditions. Ceramic/polymer composites have been investigated as weight-bearing bone grafts, but they are typically weaker than trabecular bone due to poor interfacial bonding. We hypothesized that entrapment of surface-initiated poly(ε-caprolactone) (PCL) chains on 45S5 bioactive glass (BG) particles within an in situ-formed polymer network would enhance the mechanical properties of reactive BG/polymer composites. When the surface-initiated PCL molecular weight exceeded the molecular weight between crosslinks of the network, the compressive strength of the composites increased 6- to 10-fold. The torsional strength of the composites exceeded that of human trabecular bone by a factor of two. When injected into femoral condyle defects in rats, the composites supported new bone formation at 8 weeks. The initial bone-like strength of BG/polymer composites and their ability to remodel in vivo highlight their potential for development as injectable grafts for repair of weight-bearing bone defects.

Effects of local delivery of D-amino acids from biofilm-dispersive scaffolds on infection in contaminated rat segmental defects.

Infectious complications of open fractures continue to be a significant factor contributing to non-osseous union and extremity amputation. The persistence of bacteria within biofilms despite meticulous debridement and antibiotic therapy is believed to be a major cause of chronic infection. Considering the difficulties in treating biofilm-associated infections, the use of biofilm dispersal agents as a therapeutic strategy for the prevention of biofilm-associated infections has gained considerable interest. In this study, we investigated whether local delivery of D-Amino Acids (D-AAs), a biofilm dispersal agent, protects scaffolds from contamination and reduces microbial burden within contaminated rat segmental defects in vivo. In vitro testing on biofilms of clinical isolates of Staphylococcus aureus demonstrated that D-Met, D-Phe, D-Pro, and D-Trp were highly effective at dispersing and preventing biofilm formation individually, and the effect was enhanced for an equimolar mixture of D-AAs. Incorporation of D-AAs into polyurethane scaffolds as a mixture (1:1:1 D-Met:D-Pro:D-Trp) significantly reduced bacterial contamination on the scaffold surface in vitro and within bone when implanted into contaminated femoral segmental defects. Our results underscore the potential of local delivery of d-AAs for reducing bacterial contamination by targeting bacteria within biofilms, which may represent a treatment strategy for improving healing outcomes associated with open fractures.

Pro-angiogenic and anti-inflammatory regulation by functional peptides loaded in polymeric implants for soft tissue regeneration.

Inflammation and angiogenesis are inevitable in vivo responses to biomaterial implants. Continuous progress has been made in biomaterial design to improve tissue interactions with an implant by either reducing inflammation or promoting angiogenesis. However, it has become increasingly clear that the physiological processes of inflammation and angiogenesis are interconnected through various molecular mechanisms. Hence, there is an unmet need for engineering functional tissues by simultaneous activation of pro-angiogenic and anti-inflammatory responses to biomaterial implants. In this work, the modulus and fibrinogen adsorption of porous scaffolds were tuned to meet the requirements (i.e., ~100 kPa and ~10 nm, respectively), for soft tissue regeneration by employing tyrosine-derived combinatorial polymers with polyethylene glycol crosslinkers. Two types of functional peptides (i.e., pro-angiogenic laminin-derived C16 and anti-inflammatory thymosin ß4-derived Ac-SDKP) were loaded in porous scaffolds through collagen gel embedding so that peptides were released in a controlled fashion, mimicking degradation of the extracellular matrix. The results from (1) in vitro coculture of human umbilical vein endothelial cells and human blood-derived macrophages and (2) in vivo subcutaneous implantation revealed the directly proportional relationship between angiogenic activities (i.e., tubulogenesis and perfusion capacity) and inflammatory activities (i.e., phagocytosis and F4/80 expression) upon treatment with either type of peptide. Interestingly, cotreatment with both types of peptides upregulated the angiogenic responses, while downregulating the inflammatory responses. Also, anti-inflammatory Ac-SDKP peptides reduced production of pro-inflammatory cytokines (i.e., interleukin [IL]-1ß, IL-6, IL-8, and tumor necrosis factor alpha) even when treated in combination with pro-angiogenic C16 peptides. In addition to independent regulation of angiogenesis and inflammation, this study suggests a promising approach to improve soft tissue regeneration (e.g., blood vessel and heart muscle) when inflammatory diseases (e.g., ischemic tissue fibrosis and atherosclerosis) limit the regeneration process.

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.

Characterization of the degradation mechanisms of lysine-derived aliphatic poly(ester urethane) scaffolds.

Characterization of the degradation mechanism of polymeric scaffolds and delivery systems for regenerative medicine is essential to assess their clinical applicability. Key performance criteria include induction of a minimal, transient inflammatory response and controlled degradation to soluble non-cytotoxic breakdown products that are cleared from the body by physiological processes. Scaffolds fabricated from biodegradable poly(ester urethane)s (PEURs) undergo controlled degradation to non-cytotoxic breakdown products and support the ingrowth of new tissue in preclinical models of tissue regeneration. While previous studies have shown that PEUR scaffolds prepared from lysine-derived polyisocyanates degrade faster under in vivo compared to in vitro conditions, the degradation mechanism is not well understood. In this study, we have shown that PEUR scaffolds prepared from lysine triisocyanate (LTI) or a trimer of hexamethylene diisocyanate (HDIt) undergo hydrolytic, esterolytic, and oxidative degradation. Hydrolysis of ester bonds to yield α-hydroxy acids is the dominant mechanism in buffer, and esterolytic media modestly increase the degradation rate. While HDIt scaffolds show a modest (<20%) increase in degradation rate in oxidative medium, LTI scaffolds degrade six times faster in oxidative medium. Furthermore, the in vitro rate of degradation of LTI scaffolds in oxidative medium approximates the in vivo rate in rat excisional wounds, and histological sections show macrophages expressing myeloperoxidase at the material surface. While recent preclinical studies have underscored the potential of injectable PEUR scaffolds and delivery systems for tissue regeneration, this promising class of biomaterials has a limited regulatory history. Elucidation of the macrophage-mediated oxidative mechanism by which LTI scaffolds degrade in vivo provides key insights into the ultimate fate of these materials when injected into the body.

Local delivery of tobramycin from injectable biodegradable polyurethane scaffolds.

Infections often compromise the healing of open fractures. While local antibiotic delivery from PMMA beads is an established clinical treatment of infected fractures, surgical removal of the beads is required before implanting a bone graft. A more ideal therapy would comprise a scaffold and antibiotic delivery system administered in one procedure. Biodegradable polyurethane (PUR) scaffolds have been shown in previous studies to promote new bone formation in vivo, but their potential to control infection through release of antibiotics has not been investigated. In this study, injectable PUR scaffolds incorporating tobramycin were prepared by reactive liquid molding. Scaffolds had compressive moduli of 15-115 kPa and porosities ranging from 85-93%. Tobramycin release was characterized by a 45-95% burst (tuned by the addition of PEG), followed by up to 2 weeks of sustained release, with total release 4-5-times greater than equivalent volumes of PMMA beads. Released tobramycin remained biologically active against Staphylococcus aureus, as verified by Kirby-Bauer assays. Similar results were observed for the antibiotics colistin and tigecycline. The versatility of the materials, as well as their potential for injection and controlled release, may present promising opportunities for new therapies for healing of infected wounds.