Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy.

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

Combined Administration of ASCs and BMP-12 Promotes an M2 Macrophage Phenotype and Enhances Tendon Healing.

BACKGROUND: Outcomes after intrasynovial tendon repair are highly variable. An intense inflammatory cascade followed by a delayed healing response can cause adhesion formation and repair-site failure that severely impair the function of repaired digits. No effective remedies exist to fully address these issues. Cell- and growth factor-based therapies have been shown to modulate inflammation and improve cell proliferation and matrix synthesis and therefore are promising treatment approaches for intrasynovial tendon repair. QUESTIONS/PURPOSES: (1) Can autologous adipose-derived mesenchymal stromal cells (ASCs) and recombinant bone morphogenetic protein-12 (rBMP-12) be effectively delivered to an intrasynovial flexor tendon repair without adverse effects? (2) Do autologous ASCs modulate the inflammatory response after intrasynovial tendon injury and repair? (3) Does the combined application of autologous ASCs and rBMP-12 modulate the proliferative and remodeling responses after intrasynovial tendon injury and repair? METHODS: Sixteen 1- to 2-year-old female canines were used in this study. Autologous ASC sheets, with and without rBMP-12, were applied to the surface of sutured flexor tendons. Fourteen days after repair, the effects of treatment were determined using quantitative PCR (six per group) for the expression of genes related to macrophage phenotype or inflammation (IL-4, CD163, VEGF, NOS2, IL-1B, and IFNG), cell proliferation (CCND1), and tendon formation (SCX, TNMD, COL1A1 and COL3A1). Proteomics analysis (four per group) was performed to examine changes in tendon protein abundances. CD146 immunostaining and hematoxylin and eosin staining (four per group) were used to detect tendon stem or progenitor cells and to semiquantitatively evaluate cellularity at the tendon repair; analyses were done blinded to group. RESULTS: Gross inspection and cell tracing showed that autologous ASCs and rBMP-12 were delivered to the flexor tendon repair site without the deleterious effects of adhesion and repair-site gap formation. Quantitative assessment of gene and protein expression showed effects of treatment: ASC-sheet treatment modulated the postrepair inflammatory response and facilitated healing by increasing regenerative M2 macrophages (M2 marker CD204, twofold of normal, p = 0.030), inflammatory inhibitor (prostaglandin reductase 1 [PTRG1], 1.6-fold of normal, p = 0.026), and proteins involved in tendon formation (periostin [POSTN], 1.9-fold of normal, p = 0.035). Consistently, semiquantitative and qualitative evaluations of repaired tissue showed that ASC-sheet treatment reduced mononuclear cell infiltration (12% less than nontreated tendons, p = 0.021) and introduced CD146+ stem or progenitor cells to the repair site. The combined administration of ASCs and rBMP-12 further stimulated M2 macrophages by increasing IL-4 (116-fold of normal, p = 0.002) and led to the increase of M2 effector matrix metalloproteinase-12 involved in matrix remodeling (twofold of normal, p = 0.016) and reduction of a negative regulator of angiogenesis and cell migration (StAR-related lipid transfer domain protein13 [STARD13]; 84% of normal, p = 0.000), thus facilitating the proliferative stage of tendon repair. CONCLUSIONS: ASCs and BMP-12 accelerated the progression of healing in the proliferative stage of tendon repair. The effects of ASCs and BMP-12 on tendon functional recovery should be evaluated in future studies. CLINICAL RELEVANCE: The cell sheet approach is an effective, biocompatible, and surgeon-friendly approach for cell and growth factor delivery during tendon repair. Combined application of ASCs and BMP-12 may accelerate intrasynovial tendon healing while suppressing the adverse inflammatory response.

Cell and Biologic-Based Treatment of Flexor Tendon Injuries.

The two primary factors leading to poor clinical results after intrasynovial tendon repair are adhesion formation within the digital sheath and repair-site elongation and rupture. As the outcomes following modern tendon multi-strand repair and controlled rehabilitation techniques are often unsatisfactory, alternative approaches, such as the application of growth factors and mesenchymal stem cells (MSCs), have become increasingly attractive treatment options. Successful biological therapies require carefully controlled spatiotemporal delivery of cells, growth factors, and biocompatible scaffold matrices in order to simultaneously (1) promote matrix synthesis at the tendon repair site leading to increased biomechanical strength and stiffness and (2) suppress matrix synthesis along the tendon surface and synovial sheath preventing adhesion formation. This review summarizes recent cell and biologic-based experimental treatments for flexor tendon injury, with an emphasis on large animal translational studies.

Transient, Image-Guided Gel-Dissection for Percutaneous Thermal Ablation.

Image-guided tumor ablative therapies are mainstay cancer treatment options but often require intra-procedural protective tissue displacement to reduce the risk of collateral damage to neighboring organs. Standard of care strategies, such as hydrodissection (fluidic injection), are limited by rapid diffusion of fluid and poor retention time, risking injury to adjacent organs, increasing cancer recurrence rates from incomplete tumor ablations, and limiting patient qualification. Herein, a "gel-dissection" technique is developed, leveraging injectable hydrogels for longer-lasting, shapeable, and transient tissue separation to empower clinicans with improved ablation operation windows and greater control. A rheological model is designed to understand and tune gel-dissection parameters. In swine models, gel-dissection achieves 24 times longer-lasting tissue separation dynamics compared to saline, with 40% less injected volume. Gel-dissection achieves anti-dependent dissection between free-floating organs in the peritoneal cavity and clinically significant thermal protection, with the potential to expand minimally invasive therapeutic techniques, especially across locoregional therapies including radiation, cryoablation, endoscopy, and surgery.

A hydrogel platform for co-delivery of immunomodulatory proteins for pancreatic islet allografts.

Type 1 diabetes (T1D), an autoimmune disorder in which the insulin-producing ß-cells in the islets of Langerhans in the pancreas are destroyed, afflicts over 1.6 million Americans. Although pancreatic islet transplantation has shown promise in treating T1D, continuous use of required immunosuppression regimens limits clinical islet transplantation as it poses significant adverse effects on graft recipients and does not achieve consistent long-term graft survival with 50%-70% of recipients maintaining insulin independence at 5 years. T cells play a key role in graft rejection, and rebalancing pathogenic T effector and protective T regulatory cells can regulate autoimmune disorders and transplant rejection. The synergy of the interleukin-2 (IL-2) and Fas immunomodulatory pathways presents an avenue for eliminating the need for systemic immune suppression by exploiting IL-2's role in expanding regulatory T cells and leveraging Fas ligand (FasL) activity on antigen-induced cell death of effector T cells. Herein, we developed a hydrogel platform for co-delivering an analog of IL-2, IL-2D, and FasL-presenting microgels to achieve localized immunotolerance to pancreatic islets by targeting the upregulation of regulatory T cells and effector T cells simultaneously. Although this hydrogel provided for sustained, local delivery of active immunomodulatory proteins, indefinite allograft survival was not achieved. Immune profiling analysis revealed upregulation of target regulatory T cells but also increases in Granzyme B-expressing CD8+ T cells at the graft site. We attribute the failed establishment of allograft survival to these Granzyme B-expressing T cells. This study underscores the delicate balance of immunomodulatory components important for allograft survival - whose outcome can be dependent on timing, duration, modality of delivery, and disease model.

Adhesive-based tendon-to-bone repair: failure modelling and materials selection.

Surgical reattachment of tendon to bone is a procedure marked by high failure rates. For example, nearly all rotator cuff repairs performed on elderly patients with massive tears ultimately result in recurrence of tearing. These high failure rates have been attributed to stress concentrations that arise due to the mechanical mismatch between tendon and bone. Although recent studies have identified potential adhesives with mechanical properties tuned to alleviate these stress concentrations, and thereby delay the onset of failure, resistance to the progression of failure has not been studied. Here, we refined the space of adhesive material properties that can improve surgical attachment by considering the fracture process. Using cohesive zone modelling and physiologically relevant values of mode I and mode II adhesive fracture toughnesses, we predicted the maximum displacement and strength at failure of idealized, adhesively bonded tendon-to-bone repairs. Repair failure occurred due to excessive relative displacement of the tendon and bone tissues for strong and compliant adhesives. The failure mechanism shifted to rupture of the entire repair for stiffer adhesives below a critical shear strength. Results identified a narrow range of materials on an Ashby chart that are suitable for adhesive repair of tendon to bone, including a range of elastomers and porous solids.

The effect of modified locking methods and suture materials on Zone II flexor tendon repair-An ex vivo study.

The failure rate of intrasynovial tendon repair is high due to substantial elongation at the repair site and to the development of adhesions between the tendon's surface and the surrounding digital sheath. To minimize these complications, we sought to reduce the incidence of gapping and to facilitate the initiation of early motion by improving the time zero structural properties of repair. The Winters-Gelberman 8-strand repair technique was modified by adding surface lock loops and by using Fiberwire suture material. Forty-eight canine flexor digitorum profundus tendons were transected and repaired with one of three 8-strand techniques (Pennington modified Kessler, half hitch loops, or surface locking Kessler) using either 3-0 Supramid or 4-0 Fiberwire suture. Biomechanical testing was performed to determine the physiologic and failure mode properties of the repairs. The surface locking Kessler technique improved repair maximum load, load necessary to create a 2 mm repair site gap, and yield force compared to the modified Kessler and half hitch loop techniques. Fiberwire suture improved maximum load, the load necessary to create a 2 mm repair site gap, stiffness, and yield force compared to Supramid suture. Failure occurred by both suture pull out and by suture breakage in the modified Kessler, Supramid suture repair group. Failure occurred consistently by suture breakage in the surface locking Kessler, Supramid suture repair group. These results reveal that a novel locking Kessler repair is significantly stronger than the current state-of-the art flexor tendon suture repair technique. The use of a surface locking Kessler technique with Fiberwire suture markedly improves the mechanical properties of intrasynovial tendon repair by reducing the risk of post-operative gapping and rupture.

Enhanced tendon-to-bone repair through adhesive films.

Tendon-to-bone surgical repairs have unacceptably high failure rates, possibly due to their inability to recreate the load transfer mechanisms of the native enthesis. Instead of distributing load across a wide attachment footprint area, surgical repairs concentrate shear stress on a small number of suture anchor points. This motivates development of technologies that distribute shear stresses away from suture anchors and across the enthesis footprint. Here, we present predictions and proof-of-concept experiments showing that mechanically-optimized adhesive films can mimic the natural load transfer mechanisms of the healthy attachment and increase the load tolerance of a repair. Mechanical optimization, based upon a shear lag model corroborated by a finite element analysis, revealed that adhesives with relatively high strength and low stiffness can, theoretically, strengthen tendon-to-bone repairs by over 10-fold. Lap shear testing using tendon and bone planks validated the mechanical models for a range of adhesive stiffnesses and strengths. Ex vivo human supraspinatus repairs of cadaveric tissues using multipartite adhesives showed substantial increase in strength. Results suggest that adhesive-enhanced repair can improve repair strength, and motivate a search for optimal adhesives. STATEMENT OF SIGNIFICANCE: Current surgical techniques for tendon-to-bone repair have unacceptably high failure rates, indicating that the initial repair strength is insufficient to prevent gapping or rupture. In the rotator cuff, repair techniques apply compression over the repair interface to achieve contact healing between tendon and bone, but transfer almost all force in shear across only a few points where sutures puncture the tendon. Therefore, we evaluated the ability of an adhesive film, implanted between tendon and bone, to enhance repair strength and minimize the likelihood of rupture. Mechanical models demonstrated that optimally designed adhesives would improve repair strength by over 10-fold. Experiments using idealized and clinically-relevant repairs validated these models. This work demonstrates an opportunity to dramatically improve tendon-to-bone repair strength using adhesive films with appropriate material properties.

The effect of adipose-derived stem cell sheets and CTGF on early flexor tendon healing in a canine model.

Intrasynovial tendon injuries are among the most challenging in orthopedics. Despite significant improvements in operative and rehabilitation methods, functional outcomes continue to be limited by adhesions, gap formation, and rupture. Adhesions result from excessive inflammation, whereas tendon gapping and rupture result from inflammation-induced matrix degradation and insufficient regeneration. Therefore, this study used a combined treatment approach to modulate inflammation with adipose-derived mesenchymal stromal cells (ASCs) while stimulating tendon regeneration with connective tissue growth factor (CTGF). ASCs were applied to the repair surface via cell sheets and CTGF was delivered to the repair center via porous sutures. The effect of the combined treatment was assessed fourteen days after repair in a canine flexor tendon injury model. CTGF, either alone or with ASCs, reduced inflammatory (IL1B and IL6) and matrix degrading (MMP3 and MMP13) gene expression, while increasing anti-inflammatory gene (IL4) expression and collagen synthesis compared to control repairs. The combined treatment was more effective than CTGF treatment alone, reducing the inflammatory IFNG and scar-associated COL3A1 gene expression and increasing CD146+ tendon stem/progenitor cells at the tendon surface and interior along the core suture tracks. Therefore, the combined approach is promising in promoting early flexor tendon healing and worthy of further investigation.

Effect of connective tissue growth factor delivered via porous sutures on the proliferative stage of intrasynovial tendon repair.

Recent growth factor, cell, and scaffold-based experimental interventions for intrasynovial flexor tendon repair have demonstrated therapeutic potential in rodent models. However, these approaches have not achieved consistent functional improvements in large animal trials due to deleterious inflammatory reactions to delivery materials and insufficient induction of targeted biological healing responses. In this study, we achieved porous suture-based sustained delivery of connective tissue growth factor (CTGF) into flexor tendons in a clinically relevant canine model. Repairs with CTGF-laden sutures were mechanically competent and did not show any evidence of adhesions or other negative inflammatory reactions based on histology, gene expression, or proteomics analyses at 14 days following repair. CTGF-laden sutures induced local cellular infiltration and a significant biological response immediately adjacent to the suture, including histological signs of angiogenesis and collagen deposition. There were no evident widespread biological effects throughout the tendon substance. There were significant differences in gene expression of the macrophage marker CD163 and anti-apoptotic factor BCL2L1; however, these differences were not corroborated by proteomics analysis. In summary, this study provided encouraging evidence of sustained delivery of biologically active CTGF from porous sutures without signs of a negative inflammatory reaction. With the development of a safe and effective method for generating a positive local biological response, future studies can explore additional methods for enhancing intrasynovial tendon repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2052-2063, 2018.

Enhanced Zone II Flexor Tendon Repair through a New Half Hitch Loop Suture Configuration.

This study evaluated the impact of a new half hitch loop suture configuration on flexor tendon repair mechanics. Cadaver canine flexor digitorum profundus tendons were repaired with 4- or 8-strands, 4-0 or 3-0 suture, with and without half hitch loops. An additional group underwent repair with half hitch loops but without the terminal knot. Half hitch loops improved the strength of 8-strand repairs by 21% when 4-0, and 33% when 3-0 suture was used, and caused a shift in failure mode from suture pullout to suture breakage. 8-strand repairs with half hitch loops but without a terminal knot produced equivalent mechanical properties to those without half hitch loops but with a terminal knot. 4-strand repairs were limited by the strength of the suture in all groups and, as a result, the presence of half hitch loops did not alter the mechanical properties. Overall, half hitch loops improved repair mechanics, allowing failure strength to reach the full capability of suture strength. Improving the mechanical properties of flexor tendon repair with half hitch loops has the potential to reduce the postoperative risk of gap formation and catastrophic rupture in the early postoperative period.

Surgical Sutures with Porous Sheaths for the Sustained Release of Growth Factors.

Surgical sutures with highly porous sheaths are developed using a swelling and freeze-drying procedure without compromising their mechanical properties. The modified sutures show a high capacity for loading biofactors and are able to release the loaded biofactors in a sustained manner.

The effect of mesenchymal stromal cell sheets on the inflammatory stage of flexor tendon healing.

BACKGROUND: The clinical outcomes following intrasynovial flexor tendon repair are highly variable. Excessive inflammation is a principal factor underlying the formation of adhesions at the repair surface and affecting matrix regeneration at the repair center that limit tendon excursion and impair tendon healing. A previous in-vitro study revealed that adipose-derived mesenchymal stromal cells (ASCs) modulate tendon fibroblast response to macrophage-induced inflammation. The goal of the current study was therefore to explore the effectiveness of autologous ASCs on the inflammatory stage of intrasynovial tendon healing in vivo using a clinically relevant animal model. METHODS: Zone II flexor tendon transections and suture repairs were performed in a canine model. Autologous ASC sheets were delivered to the surface of repaired tendons. Seven days after repair, the effects of ASCs on tendon healing, with a focus on the inflammatory response, were evaluated using gene expression assays, immunostaining, and histological assessments. RESULTS: ASCs delivered via the cell sheet infiltrated the host tendon, including the repair surface and the space between the tendon ends, as viewed histologically by tracking GFP-expressing ASCs. Gene expression results demonstrated that ASCs promoted a regenerative/anti-inflammatory M2 macrophage phenotype and regulated tendon matrix remodeling. Specifically, there were significant increases in M2-stimulator (IL-4), marker (CD163 and MRC1), and effector (VEGF) gene expression in ASC-sheet treated tendons compared with nontreated tendons. When examining changes in extracellular matrix expression, tendon injury caused a significant increase in scar-associated COL3A1 expression and reductions in COL2A1 and ACAN expression. The ASC treatment effectively counteracted these changes, returning the expression levels of these genes closer to normal. Immunostaining further confirmed that ASC treatment increased CD163+ M2 cells in the repaired tendons and suppressed cell apoptosis at the repair site. CONCLUSIONS: This study provides a novel approach for delivering ASCs with outcomes indicating potential for substantial modulation of the inflammatory environment and enhancement of tendon healing after flexor tendon repair.

Tunability of collagen matrix mechanical properties via multiple modes of mineralization.

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering.

Shear lag sutures: Improved suture repair through the use of adhesives.

Suture materials and surgical knot tying techniques have improved dramatically since their first use over five millennia ago. However, the approach remains limited by the ability of the suture to transfer load to tissue at suture anchor points. Here, we predict that adhesive-coated sutures can improve mechanical load transfer beyond the range of performance of existing suture methods, thereby strengthening repairs and decreasing the risk of failure. The mechanical properties of suitable adhesives were identified using a shear lag model. Examination of the design space for an optimal adhesive demonstrated requirements for strong adhesion and low stiffness to maximize the strength of the adhesive-coated suture repair construct. To experimentally assess the model, we evaluated single strands of sutures coated with highly flexible cyanoacrylates (Loctite 4903 and 4902), cyanoacrylate (Loctite QuickTite Instant Adhesive Gel), rubber cement, rubber/gasket adhesive (1300 Scotch-Weld Neoprene High Performance Rubber & Gasket Adhesive), an albumin-glutaraldehyde adhesive (BioGlue), or poly(dopamine). As a clinically relevant proof-of-concept, cyanoacrylate-coated sutures were then used to perform a clinically relevant flexor digitorum tendon repair in cadaver tissue. The repair performed with adhesive-coated suture had significantly higher strength compared to the standard repair without adhesive. Notably, cyanoacrylate provides strong adhesion with high stiffness and brittle behavior, and is therefore not an ideal adhesive for enhancing suture repair. Nevertheless, the improvement in repair properties in a clinically relevant setting, even using a non-ideal adhesive, demonstrates the potential for the proposed approach to improve outcomes for treatments requiring suture fixation. Further study is necessary to develop a strongly adherent, compliant adhesive within the optimal design space described by the model.

Biochemical and physical signal gradients in hydrogels to control stem cell behavior.

Three-dimensional (3D) gradients of biochemical and physical signals in macroscale degradable hydrogels are engineered that can regulate photoencapsulated human mesenchymal stem cell (hMSC) behavior. This simple, cytocompatible, and versatile gradient system may be a valuable tool for researchers in biomaterials science to control stem cell fate in 3D and guide tissue regeneration.

A rapid protein folding assay for the bacterial periplasm.

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N-terminal signal recognition particle (SRP)-dependent signal peptide and a C-terminal selectable marker, TEM-1 beta-lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo beta-lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding-enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.

Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli.

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin-arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N-terminal Tat export signal and a C-terminal selectable marker, namely beta-lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.