Kim S. Jones

Effects of biomaterial-induced inflammation on fibrosis and rejection.

Evidence is emerging that biomaterials cause inflammation by ligating innate immune receptors on antigen presenting cells. Although inflammation is usually viewed as detrimental, it has unexpected and potentially beneficial effects on fibrosis and transplant rejection. For example, the magnitude of inflammation due to a biomaterial is not predictive of the extent of fibrosis. Similarly, biomaterials do not always show adjuvancy. Some biomaterials suppressed T cell rejection responses in vivo and in vitro, while others non-specifically stimulated T cell proliferation. Understanding these complex inter-relationships is the key to designing a biomaterial that stimulates regeneration and induces tolerance in tissue engineering applications.

Effect of alginate on innate immune activation of macrophages

Alginate, a natural polysaccharide, has been widely used in tissue engineering and drug delivery, but like other biomaterials, it causes inflammation by unknown mechanisms. We hypothesized that alginate would stimulate innate immune responses through macrophage receptors. In this study, we showed that sodium alginate induced activation of macrophage-like cells (RAW264.7) through the NF-kappaB pathway. Production of proinflammatory cytokines, such as IL-1beta, IL-6, IL-12, and TNF-alpha was time and dose-dependent. Treatment with alginate solution caused responses that closely paralleled stimulation by lipopolysaccharide in timing and magnitude. These data suggest that sodium alginate causes innate immune responses through NF-kappaB activation and likely activates the same pathways as pathogen recognition.

Carbohydrate composition and freezing tolerance of canes and buds in Vitis vinifera

Summary In grapevines, buds and canes have different strategies of freezing tolerance - buds avoid freezing by supercooling, whereas canes tolerate extracellular freezing. To further understand the relationship between freezing tolerance and carbohydrate composition of these tissues in Vitis vinifera, analyses were made of starch, glucose, fructose, sucrose and raffinose of field-grown vines during natural acclimation and deacclimation over two years, on artificially deacclimated field material, and on daylength induced dormant growthroom material. Statistics on correlation and path coefficients were calculated to detect relationships, and a was set at 0.05. During the acclimation and deacclimation of grapevines in the field, the level of most carbohydrates was correlated with freezing tolerance in both buds and canes. Starch levels were negatively correlated with hardiness whereas most soluble sugars were positively correlated. Artificial deacclimation with heat treatment supported these relationships between LTSo and carbohydrates. Dormancy induction by short day treatment did not increase freezing tolerance but did increase raffinose in buds. Statistical path analysis highlighted the importance of starch and fructose in freezing tolerance of grapevines, and indicated that the different soluble sugars may have different roles in buds and canes. This statistical method holds great potential, particularly for identifying transformation targets from data on metabolites.

Biomaterials as vaccine adjuvants

Biomaterials are promising candidate adjuvants to enhance vaccine efficacy. Through adjuvant design, we can broaden the use of vaccines to diseases such as AIDS, malaria, and cancer. This review addresses the fundamentals of vaccine and adjuvant function in order to determine guidelines for adjuvant design, including aspects of the vaccine such as disease target, antigen formulation, and delivery route. An ideal biomaterial adjuvant will perform three functions. (1) It will deliver the antigen selectively to dendritic cells. This has been accomplished through release of chemokines or cytokines, use of antidendritic cell antibodies, and even through particle size selection. (2) It will activate the dendritic cells, improving antigen presentation. Biomaterials themselves have been shown to activate innate immunity, but specific innate‐activating ligands have also been included in adjuvant formulations. Finally, (3) it will release the antigen appropriately into the dendritic cell. Tuning release to be pH sensitive and engineering endosomal release are strategies that have been used. There is a real opportunity to rationally design better biomaterial adjuvants that will significantly expand and improve vaccine function.

Dendrimer‐grafted cell adhesion peptide–modified PDMS

Surface concentration of cell adhesion peptides is thought to play a role in the interactions between biomaterials and cells. The high density of functional groups at the periphery of dendrimers has been exploited in various applications, but their full potential for generating surfaces with high functional group concentrations has not yet been realized. Poly(dimethylsiloxane) elastomers were surface modified with both polyethylene oxide (PEO) and generation 3 diaminobutane dendrimers. PEO and the dendrimers were subsequently used as linker molecules for surface grafting of cell adhesion peptides. ATR‐FTIR, X‐ray photoelectron spectroscopy, and water contact angle results confirmed the successful attachment of the polymer linkers and peptides. Peptide grafting density was quantified by means of 125I radiolabeling. Maximum surface peptide grafting density on dendrimer‐modified surfaces was twofold greater than the maximum peptide grafting density achieved via the PEO linker. However, vascular endothelial cell adhesion was significantly greater on surfaces modified with the PEO linker, presumably due to the highly flexible PEO spacer making the peptide more accessible for binding with the cell surface receptors. These results suggest that, although peptide surface density may be important, optimizing surface density may not be sufficient for improving biological interactions.

The recognition of biomaterials: Pattern recognition of medical polymers and their adsorbed biomolecules

All biomedical materials are recognized as foreign entities by the host immune system despite the substantial range of different materials that have been developed by material scientists and engineers. Hydrophobic biomaterials, hydrogels, biomaterials with low protein binding surfaces, and those that readily adsorb a protein layer all seem to incite similar host responses in vivo that may differ in magnitude, but ultimately result in encapsulation by fibrotic tissue. The recognition of medical materials by the host is explained by the very intricate pattern recognition system made up of integrins, toll-like receptors, scavenger receptors, and other surface proteins that enable leukocytes to perceive almost any foreign body. In this review, we describe the various pattern recognition receptors and processes that occur on biomedical material surfaces that permit detection of a range of materials within the host.

Characterization of viability and proliferation of alginate-poly-L-lysine–alginate encapsulated myoblasts using flow cytometry

Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

Suppressed Splenocyte Proliferation Following a Xenogeneic Skin Graft due to Implanted Biomaterials

Background. Immune system responses to antigens in the context of biomaterials are poorly understood. Biomaterial implantation results in an inflammatory reaction, which is anticipated to alter the adaptive immune response, in the case presented here, to a skin xenograft. Our earlier work showed unexpectedly low splenocyte proliferation following a xenogeneic cell implant in tandem with a biomaterial in the form of a microcapsule. Here we explore whether that effect was due to the cells or the biomaterial, and attempt to dissect the mechanism of immune deviation. Methods. We assessed the immune response of Balb/c mice to hamster skin grafts accompanied by one of three implants: encapsulated xenogeneic cells; free cells accompanied by the same encapsulation biomaterials; and the encapsulation biomaterials without cells. Cells were encapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer then embedded in an agarose gel. Splenocyte proliferation upon re-challenge in vitro, antibody titer in serum, and Th1/2 polarization (by cytokines in splenocyte challenge supernatants and antibody isotypes in serum) were measured. Results. All skin grafts with encapsulation materials (even without cells) suppressed subsequent splenocyte proliferation at 10 days postimplant, athough this effect disappeared by two months. In contrast, the antibody response was equal to or greater than that for a skin graft alone. Th1/2 polarization could not explain these observations because it did not correlate with the suppression of splenocyte proliferation. Conclusions. Implanted biomaterials caused nonspecific, transient suppression of splenocyte responses to hamster cells following hamster skin grafts, which is potentially important in the context of tissue engineering.

Transient inhibition of connective tissue infiltration and collagen deposition into porous poly(lactic-co-glycolic acid) discs.

Connective tissue rapidly proliferates on and around biomaterials implanted in vivo, which impairs the function of the engineered tissues, biosensors, and devices. Glucocorticoids can be utilized to suppress tissue ingrowth, but can only be used for a limited time because they nonselectively arrest cell proliferation in the local environment. The present study examined use of a prolyl-4-hydroxylase inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), to suppress connective tissue ingrowth in porous PLGA discs implanted in the peritoneal cavity for 28 days. The prolyl-4-hydroxylase inhibitor was found to be effective at inhibiting collagen deposition within and on the outer surface of the disc, and also limited connective tissue ingrowth, but not to the extent of glucocorticoid inhibition. Finally, it was discovered that 1,4-DPCA suppressed Scavenger Receptor A expression on a macrophage-like cell culture, which may account for the drugs ability to limit connective tissue ingrowth in vivo.

Retroviral expression of MIR2 decreases both surface MHC class I and the alloimmune CTL response

The immune response to allogeneic cells in tissue‐engineered constructs is a major barrier to their successful application in the treatment of many human diseases. Specifically, the T cell‐mediated immune response, initiated through the recognition of cell surface MHCI molecules, is the primary cause of acute cellular allograft rejection. In this study, we altered expression of MHCI through viral immunomodulatory mechanisms to examine whether allogeneic cells could be made to ‘mimic’ viral evasion of a host CTL response. We demonstrate the successful application of a retroviral vector in vitro to overexpress the Kaposis sarcoma‐associated herpesvirus immunomodulatory protein, MIR2, in human monocyte‐like myeloid progenitor cells. This approach led to differential downregulation of cell surface MHCI, ICAM‐1 and B7–2 molecules. We also demonstrate that downregulation of immunoactive molecules has the functional effect of significantly reducing T cell‐mediated cytotoxicity without altering NK‐mediated cytotoxicity in vitro. These results provide proof‐of‐concept that viral immune evasion strategies allow cell‐based tissue‐engineered constructs to delay or even prevent acute cellular immune rejection in vivo. Importantly, this methodology could facilitate the development of universal donor cells for tissue engineering applications. Copyright