Biomaterials-based immunomodulation enhances survival of murine vascularized composite allografts.

Vascularized composite allotransplantation (VCA) is a restorative option for patients suffering from severe tissue defects not amenable to conventional reconstruction. However, the toxicities associated with life-long multidrug immunosuppression to enable allograft survival and induce immune tolerance largely limit the broader application of VCA. Here, we investigate the potential of targeted immunomodulation using CTLA4-Ig combined with a biological porcine-derived extracellular matrix (ECM) scaffold that elicits a pro-regenerative Th2 response to promote allograft survival and regulate the inflammatory microenvironment in a stringent mouse orthotopic hind limb transplantation model (BALB/c to C57BL/6). The median allograft survival time (MST) increased significantly from 15.0 to 24.5 days (P = 0.0037; Mantel-Cox test) after adding ECM to the CTLA4-Ig regimen. Characterization of the immune infiltration shows a pro-regenerative phenotype prevails over those associated with inflammation and rejection including macrophages (F4/80hi+CD206hi+MHCIIlow), eosinophils (F4/80lowSiglec-F+), and T helper 2 (Th2) T cells (CD4+IL-4+). This was accompanied by an increased expression of genes associated with a Type 2 polarized immune state such as Il4, Ccl24, Arg1 and Ym1 within the graft. Furthermore, when ECM was applied along with a clinically relevant combination of CTLA4-Ig and Rapamycin, allograft survival was prolonged from 33.0 to 72.5 days (P = 0.0067; Mantel-Cox test). These studies implicate the clinical exploration of combined regimens involving local application of pro-regenerative, immunomodulatory biomaterials in surgical wound sites with targeted co-stimulatory blockade to reduce adverse effects of immunosuppression and enhance graft survival in VCA.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

Autologous Protein Solution processing alters lymphoid and myeloid cell populations and modulates gene expression dependent on cell type.

Osteoarthritis (OA) is a degenerative disease associated with cartilage degradation, osteophyte formation, and fibrillation. Autologous Protein Solution (APS), a type of autologous anti-inflammatory orthobiologic, is used for pain management and treatment of OA. Various compositions of autologous PRP formulations are in clinical use for musculoskeletal pathologies, by nature of their minimal processing and source of bioactive molecules. Currently, there is no consensus on the optimal composition of the complex mixture. In this study, we focused on elucidating the immune cell subtypes and phenotypes in APS. We identified the immune cell types in APS from healthy donors and investigated phenotypic changes in the immune cells after APS processing. Based on flow cytometric analysis, we found that neutrophils and T cells are the most abundant immune cell types in APS, while monocytes experience the largest fold change in concentration compared to WBCs. Gene expression profiling revealed that APS processing results in differential gene expression changes dependent on immune cell type, with the most significantly differentially regulated genes occurring in the monocytes. Our results demonstrate that the mechanical processing of blood, whose main purpose is enrichment and separation, can alter its protein and cellular composition, as well as cellular phenotypes in the final product.

Role of Inflammation and the Immune System in the Progression of Osteoarthritis.

Understanding the molecular drivers and feedback loops of osteoarthritis (OA) may provide future therapeutic strategies to modulate the disease progression. The current paradigm of OA is evolving from a purely mechanical disease caused by cartilage wear toward a complex biological response connecting biomechanics, inflammation, and the immune system. The view of OA as a chronic wound highlights the role inflammation plays and also the body's attempts to repair an ongoing injury. Inflammatory signals, including cytokines such as interleukin-1 and tissue necrosis factor α, surface-expressed pattern recognition receptors such as toll-like receptors 2 and 4, complement factors such as C5, as well as pathogen-associated molecular patterns and damage-associated molecular patterns drive the enzymatic cascade that degrades cartilage matrix in OA. Considering the joint as an entire organ, interactions between the cells that reside in the synovium including macrophages and other immune cells, appear to drive enzymatic activity in cartilage, which, in turn, feeds signals back to the synovium that continues stimulating degradation in a feed-forward loop. This review will explore the potential roles of immune cells such as macrophages and T cells in the synovium in both stimulating and modulating the inflammatory response in OA. © 2019 Orthopedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:253-257, 2020.

Interleukin-36γ-producing macrophages drive IL-17-mediated fibrosis.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)-4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17-dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

Temperature-Activated PEG Surface Segregation Controls the Protein Repellency of Polymers.

Poly(ethylene glycol) (PEG) is widely used to modulate the hydration states of biomaterials and is often applied to produce nonfouling surfaces. Here, we present X-ray scattering data, which show that it is the surface segregation of PEG, not just its presence in the bulk, that makes this happen by influencing the hydrophilicity of PEG-containing substrates. We demonstrate a temperature-dependent trigger that transforms a PEG-containing substrate from a protein-adsorbing to a protein-repelling state. On films of poly(desaminotyrosyl-tyrosine-co-PEG carbonate) with high (20 wt %) PEG content, in which very little protein adsorption is expected, quartz crystal microbalance data showed significant adsorption of fibrinogen and bovine serum albumin at 8 °C. The surface became protein-repellent at 37.5 °C. When the same polymer was iodinated, the polymer was protein-adsorbent, even when 37 wt % PEG was incorporated into the polymer backbone. This demonstrates that high PEG content by itself is not sufficient to repel proteins. By inhibiting phase separation either with iodine or by lowering the temperature, we show that PEG must phase-separate and bloom to the surface to create an antifouling surface. These results suggest an opportunity to design materials with high PEG content that can be switched from a protein-attractant to a protein-repellent state by inducing phase separation through brief exposure to temperatures above their glass transition temperature.

A hyaluronic acid binding peptide-polymer system for treating osteoarthritis.

Hyaluronic acid (HA) is found naturally in synovial fluid and is utilized therapeutically to treat osteoarthritis (OA). Here, we employed a peptide-polymer cartilage coating platform to localize HA to the cartilage surface for the purpose of treating post traumatic osteoarthritis. The objective of this study was to increase efficacy of the peptide-polymer platform in reducing OA progression in a mouse model of post-traumatic OA without exogenous HA supplementation. The peptide-polymer is composed of an HA-binding peptide (HABP) conjugated to a heterobifunctional poly (ethylene glycol) (PEG) chain and a collagen binding peptide (COLBP). We created a library of different peptide-polymers and characterized their HA binding properties in vitro using quartz crystal microbalance (QCM-D) and isothermal calorimetry (ITC). The peptide polymers were further tested in vivo in an anterior cruciate ligament transection (ACLT) murine model of post traumatic OA. The peptide-polymer with the highest affinity to HA as tested by QCM-D (∼4-fold greater binding compared to other peptides tested) and by ITC (∼3.8-fold) was HABP2-8-arm PEG-COLBP. Biotin tagging demonstrated that HABP2-8-arm PEG-COLBP localizes to both cartilage defects and synovium. In vivo, HABP2-8-arm PEG-COLBP treatment and the clinical HA comparator Orthovisc lowered levels of inflammatory genes including IL-6, IL-1B, and MMP13 compared to saline treated animals and increased aggrecan expression in young mice. HABP2-8-arm PEG-COLBP and Orthovisc also reduced pain as measured by incapacitance and hotplate testing. Cartilage degeneration as measured by OARSI scoring was also reduced by HABP2-8-arm PEG-COLBP and Orthovisc. In aged mice, HABP2-8-arm PEG-COLBP therapeutic efficacy was similar to its efficacy in young mice, but Orthovisc was less efficacious and did not significantly improve OARSI scoring. These results demonstrate that HABP2-8-arm PEG-COLBP is effective at reducing PTOA progression.

Cyclodextrin Modulated Type I Collagen Self-Assembly to Engineer Biomimetic Cornea Implants.

Collagen-rich tissues in the cornea exhibit unique and highly organized extracellular matrix ultrastructures, which contribute to its high load-bearing capacity and light transmittance. Corneal collagen fibrils are controlled during development by small leucine-rich proteoglycans (SLRPs) that regulate the fibril diameter and spacing in order to achieve the unique optical transparency. Cyclodextrins (CDs) of varying size and chemical functionality for their ability to regulate collagen assembly during vitrification process are screened in order to create biosynthetic materials that mimic the native cornea structure. Addition of ßCD to collagen vitrigels produces materials with aligned fibers and lamellae similar to native cornea, resulting in mechanically robust and transparent materials. Biochemistry analysis revealed that CD interacts with hydrophobic amino acids in collagen to influence assembly and fibril organization. To translate the self-assembled collagen materials for cornea reconstruction, custom molds for gelation and vitrification are engineered to create ßCD/Col implants with curvature matching that of the cornea. Acellular ßCD/Col materials are implanted in a rabbit partial keratoplasty model with interrupted sutures. The implants demonstrate tissue integration and support re-epithelialization. Therefore, the addition of CD molecules regulates collagen self-assembly and provides a simple process to engineer corneal mimetic substitutes with advanced structural and functional properties.

Proteomic composition and immunomodulatory properties of urinary bladder matrix scaffolds in homeostasis and injury.

Urinary bladder matrix (UBM) is used clinically for management of wounds and reinforcement of surgical soft tissue repair, among other applications. UBM consists of the lamina propria and basal lamina of the porcine urinary bladder, and is decellularized as part of the process to manufacture the medical device. UBM is composed mainly of Collagen I, but also contains a wide variety of fibrillar and basement membrane collagens, glycoproteins, proteoglycans and ECM-associated factors. Upon application of the biomaterial in a traumatic or non-traumatic setting in a mouse model, there is a cascade of immune cells that respond to the damaged tissue and biomaterial. Here, through the use of multicolor flow cytometry, we describe the various cells that infiltrate the UBM scaffold in a subcutaneous and volumetric muscle injury model. A wide variety of immune cells are found in the UBM scaffold immune microenvironment (SIM) including F4/80+ macrophages, CD11c+ dendritic cells, CD3+ T cells and CD19+ B cells. A systemic IL-4 upregulation and a local M2-macrophage response were observed in the proximity of the implanted UBM. The recruitment and activation of these cells is dependent upon signals from the scaffold and communication between the different cell types present.

Targeted delivery of hyaluronic acid to the ocular surface by a polymer-peptide conjugate system for dry eye disease.

Hyaluronic acid (HA) solutions effectively lubricate the ocular surface and are used for the relief of dry eye related symptoms. However, HA undergoes rapid clearance due to limited adhesion, which necessitates frequent instillation. Conversely, highly viscous artificial tear formulations with HA blur vision and interfere with blinking. Here, we developed an HA-eye drop formulation that selectively binds and retains HA for extended periods of time on the ocular surface. We synthesized a heterobifunctional polymer-peptide system with one end binding HA while the other end binding either sialic acid-containing glycosylated transmembrane molecules on the ocular surface epithelium, or type I collagen molecule within the tissue matrix. HA solution was mixed with the polymer-peptide system and tested on both ex vivo and in vivo models to determine its ability to prolong HA retention. Furthermore, rabbit ocular surface tissues treated with binding peptides and HA solutions demonstrated superior lubrication with reduced kinetic friction coefficients compared to tissues treated with conventional HA solution. The results suggest that binding peptide-based solution can keep the ocular surface enriched with HA for prolonged times as well as keep it lubricated. Therefore, this system can be further developed into a more effective treatment for dry eye patients than a standard HA eye drop. STATEMENT OF SIGNIFICANCE: Eye drop formulations containing HA are widely used to lubricate the ocular surface and relieve dry eye related symptoms, however its low residence time remains a challenge. We designed a polymer-peptide system for the targeted delivery of HA to the ocular surface using sialic acid or type I collagen as anchors for HA immobilization. The addition of the polymer-peptide system to HA eye drop exhibited a reduced friction coefficient, and it can keep the ocular surface enriched with HA for prolonged time. This system can be further developed into a more effective treatment for dry eye than a standard HA eye drop.

Competitive Adsorption of Plasma Proteins Using a Quartz Crystal Microbalance.

Proteins that get adsorbed onto the surfaces of biomaterials immediately upon their implantation mediate the interactions between the material and the environment. This process, in which proteins in a complex mixture compete for adsorption sites on the surface, is determined by the physicochemical interactions at the interface. Competitive adsorption of bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I), sequentially and from mixtures, was investigated so as to understand the performances of different surfaces used in biomedical applications. A quartz crystal microbalance with dissipation was used to monitor the adsorption of these proteins onto two materials used in functional bone replacement, a titanium alloy (Ti6Al4V) and Ti6Al4V physisorbed with poly(sodium styrenesulfonate) [poly(NaSS)], and three controls, gold, poly(desaminotyrosyltyrosine ethyl ester carbonate) [poly(DTEc)], and polystyrene (PS). In experiments with individual proteins, the adsorption was the highest with Fn and Col I and the least with BSA. Also, protein adsorption was the highest on poly(NaSS) and Ti6Al4V and the least on poly(DTEc). In sequential adsorption experiments, protein exchange was observed in BSA + Fn, Fn + Col I, and BSA + Col I sequences but not in Fn + BSA and Col I + BSA because of the lower affinity of BSA to surfaces relative to Fn and Col I. Protein adsorption was the highest with Col I + Fn on hydrophobic surfaces. In experiments with protein mixtures, with BSA & Fn, Fn appears to be preferentially adsorbed; with Fn & Col I, both proteins were adsorbed, probably as multilayers; and with Col I & BSA, the total amount of protein was the highest, greater than that in sequential and individual adsorption of the two proteins, probably because of the formation of BSA and Col I complexes. Protein conformational changes induced by the adsorbing surfaces, protein-protein interactions, and affinities of proteins appear to be the important factors that govern competitive adsorption. The findings reported here will be useful in understanding the host response to surfaces used for implants.

Time to Relax: Mechanical Stress Release Guides Stem Cell Responses.

Stem cells integrate spatiotemporal cues, including the mechanical properties of their microenvironment, into their fate decisions. Chaudhuri et al. (2015) show that the ability of the extracellular matrix to dissipate cell-induced forces, referred to as stress-relaxation, is a key mechanical signal influencing stem cell fate and function.

Poly(NaSS) functionalization modulates the conformation of fibronectin and collagen type I to enhance osteoblastic cell attachment onto Ti6Al4V.

Functionalization of surfaces with poly(sodium styrenesulfonate) (poly(NaSS)) has recently been found to enhance osteointegration of implantable materials. Radical polymerization of poly(NaSS) on titanium (Ti)-based substrates has been used to improve their long-term performance by preventing fibrosis and consequently implant loosening. However, the influence of the sulfonate groups on the early cell behavior and the associated molecular phenomena remains to be understood. In this work, we used quartz crystal microbalance with dissipation (QCM-D) to elucidate the role of poly(NaSS) in enhancing osteoblastic cell attachment. This was measured by following the cell attachment using the MC3T3-E1 cell line, on fetal bovine serum (FBS) preadsorbed surfaces and on substrates adsorbed with a series of relevant proteins, bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I). Comparison of the performance of poly(NaSS) with other clinically important substrates such as Ti alloy Ti6Al4V, gold, and poly(desamino-tyrosyl-tyrosine ethyl ester carbonate) (poly(DTEc)) indicates poly(NaSS) to be a superior substrate for MC3T3-E1 cells attachment. This attachment was found to be integrin mediated in the presence of Fn and Col I. Antibodies specific to the RGD peptide and the N- and C-terminal HB-binding domains reacted more intensively with Fn adsorbed on poly(NaSS). Fn adapts a conformation favorable to RGD mediated cell attachment when adsorbed onto poly(NaSS).

Enzymatic surface erosion of high tensile strength polycarbonates based on natural phenols.

Surface erosion has been recognized as a valuable design tool for resorbable biomaterials within the context of drug delivery devices, surface coatings, and when precise control of strength retention is critical. Here we report on high tensile strength, aromatic-aliphatic polycarbonates based on natural phenols, tyrosol (Ty) and homovanillyl alcohol (Hva), that exhibit enzymatic surface erosion by lipase. The Young's moduli of the polymers for dry and fully hydrated samples are 1.0 to 1.2 GPa and 0.8 to 1.2 GPa, respectively. Typical characteristics of enzymatic surface erosion were confirmed for poly(tyrosol carbonate) films with concomitant mass-loss and thickness-loss at linear rates of 0.14 ± 0.01 mg cm(-2) d(-1) and 3.0 ± 0.8 µm d(-1), respectively. The molecular weight and the mechanical properties of the residual films remained constant. Changing the ratio of Ty and Hva provided control over the glass transition temperature (T(g)) and the enzymatic surface erosion: increasing the Hva content in the polymers resulted in higher T(g) and lower enzymatic erosion rate. Polymers with more than 50 mol % Hva were stable at 37 °C in enzyme solution. Analysis on thin films using quartz crystal microbalance with dissipation (QCM-D) demonstrated that the onset temperature of the enzymatic erosion was approximately 20 °C lower than the wet T(g) for all tested polymers. This new finding demonstrates that relatively high tensile strength polycarbonates can undergo enzymatic surface erosion. Moreover, it also sheds light on the connection between T(g) and enzymatic degradation and explains why few of the high strength polymers follow an enzyme-meditated degradation pathway.

Oriented, multimeric biointerfaces of the L1 cell adhesion molecule: an approach to enhance neuronal and neural stem cell functions on 2-D and 3-D polymer substrates.

This article focuses on elucidating the key presentation features of neurotrophic ligands at polymer interfaces. Different biointerfacial configurations of the human neural cell adhesion molecule L1 were established on two-dimensional films and three-dimensional fibrous scaffolds of synthetic tyrosine-derived polycarbonate polymers and probed for surface concentrations, microscale organization, and effects on cultured primary neurons and neural stem cells. Underlying polymer substrates were modified with varying combinations of protein A and poly-D-lysine to modulate the immobilization and presentation of the Fc fusion fragment of the extracellular domain of L1 (L1-Fc). When presented as an oriented and multimeric configuration from protein A-pretreated polymers, L1-Fc significantly increased neurite outgrowth of rodent spinal cord neurons and cerebellar neurons as early as 24 h compared to the traditional presentation via adsorption onto surfaces treated with poly-D-lysine. Cultures of human neural progenitor cells screened on the L1-Fc/polymer biointerfaces showed significantly enhanced neuronal differentiation and neuritogenesis on all protein A oriented substrates. Notably, the highest degree of ßIII-tubulin expression for cells in 3-D fibrous scaffolds were observed in protein A oriented substrates with PDL pretreatment, suggesting combined effects of cell attachment to polycationic charged substrates with subcellular topography along with L1-mediated adhesion mediating neuronal differentiation. Together, these findings highlight the promise of displays of multimeric neural adhesion ligands via biointerfacially engineered substrates to "cooperatively" enhance neuronal phenotypes on polymers of relevance to tissue engineering.