Immune response testing of electrospun polymers: an important consideration in the evaluation of biomaterials

Immune Response Testing of Electrospun Polymers: An Important Consideration in the Evaluation of Biomaterials Matthew J. Smith1, Donna C. Smith2, Kimber L. White, Jr.2, and Gary L. Bowlin1 1Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia USA 2Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia USA Disclosure: Gary L. Bowlin has United States and International patents pending concerning technology presented in this
manuscript, and this technology has been licensed to NanoMatrix, Inc., in which he has a financial interest. Kimber L. White, Jr.
is the owner of a company, ImmunoTox, Inc., that conducts immunotoxicological studies under GLP, however none of the work
presented here involved his company.

ABSTRACT
the immune system lack standardization, and they Due primarily to cell sourcing issues, many in the often only evaluate one small component of the field of tissue engineering have opted to create immune response: inflammation. Depending on the scaffolds that promote in situ regeneration, using the objective of the study, this approach may or may not body as both the bioreactor and the cell source for the be sufficient for evaluating immunocompetance remodeling of scaffolds, resulting in the formation of following exposure to a material. Often not native tissue. This practice raises many concerns, evaluated are effects of biomaterials on acquired with the body’s immune response to such an implant (cell-mediated, i.e. T-cell; and humoral, i.e. B-cell) often being neglected as a potential problem in immunity or other innate parameters, such as Natural preliminary design and biocompatibility testing. Killer (NK) cell activity. In addition, the responses More importantly, what happens over time in terms of both innate and acquired immunity to electrospun of the immune responses as the biodegradable biomaterials have been completely ignored. It is scaffold structures being utilized to promote in situ imperative that an overall assessment of regeneration begin to degrade, forming structural immunomodulation be acquired for a given fragments and degradation products? In summary, biomaterial before embarking on the more detailed, immune response evaluations are critical mechanistic approaches that pervade biomaterial considerations that must be conducted when research today. The approach utilized by one of this evaluating bioresorbable scaffolds. In addition, it is paper’s coauthors (White, [2-9]) to evaluate essential that these evaluations analyze materials for biomaterials illustrates a comprehensive their potential dose-response and time-course effects methodology to evaluate the multiple components of on the various components of innate and acquired the immune response. In addition, this approach provides a unique opportunity to advance the study of the immune responses to a wide variety of EDITORIAL BODY
biomaterials and allows for the standardization of a Research into the immune response to biomaterials testing hierarchy for evaluating the complete host has been indicated as essential to a complete immune response (both innate and adaptive evaluation of their biocompatibility [1]. Recent work immunity) following exposure to biomaterials. examining the host response to biomaterials has provided some insight into the inflammatory The immune response to a biomaterial is not unlike response to these materials. Routine evaluations of the reaction to any other foreign substance. Highly biomaterials often consist of co-culturing of complex and involving many different cell types, macrophages and/or L929 mouse fibroblasts with the both innate and acquired immune responses are material, followed by examination of various initiated when increased levels of chemokines indicators of inflammation. To many in the (chemoattractant signals) result in the initiation of the biomedical engineering community, this information inflammatory response, eliciting the infiltration of is the “gold standard” by which a material is deemed phagocytic cells, including polymorphonuclear cells to elicit an acceptable (or undesirable) immune (PMN) and macrophages. In addition, biomaterial response. Current approaches in biomaterials surface contact with the complement protein research that investigate the effects of biomaterials on fragment C3b (produced continuously at low levels) Journal of Engineered Fibers and Fabrics activates the alternate pathway of the complement surface chemistry (hydrophilicity and surface charge) cascade. By-products of this cascade (i.e. C5a, C3b, of the biomaterial evaluated modulates expression of and iC3b) can promote macrophage activation and anti-inflammatory or pro-inflammatory cytokines opsonization [10, 11]; macrophages can be stimulated to release a wide variety of signals (cytokines), including interleukins (IL), interferons (IFN), and Others have focused specifically on the macrophage tumor necrosis factors (TNF), which in turn mediate and its interactions with biomaterials. Greisler et al. other processes, including maintaining inflammation, have published extensively regarding macrophage promoting T- and B-cell development, chemotaxis, interactions with synthetic bioresorbable materials, and activation, and initiating tissue repair (collagen demonstrating that macrophage contact with these production) and angiogenesis [12]. Upon activation, materials promotes activation of the macrophage, T- and B-cells (lymphocytes) respond to foreign followed by biomaterial-specific release of growth antigens, and while these responses are usually factors and various cytokines [20]. In addition, they slower than innate responses, they are highly antigen- reported that, when cultured in media preconditioned specific, in contrast to innate immune responses. by macrophage-polymer interactions, endothelial Known as adaptive immunity, these cells are capable cells demonstrated increased 3H-thymidine of proliferation when appropriately stimulated, and incorporation with increasing macrophage-material they are highly adept at discriminating self from non- interaction time [21]. Macrophages have also been self. Additionally, lymphocytes are capable of shown to promote endothelialization following developing a memory response, and in the event of a exposure to vascular prosthetics composed of second exposure to the antigen, the immune response bioresorbable materials, where the rate of endothelial is more rapid and of a higher magnitude. In cell ingrowth is directly related to the rate of particular, both the secondary antibody response (humoral) and the delayed-type hypersensitivity response (cell-mediated) quickly produce activated Perhaps the most extensively investigated biomaterial effector cells in larger numbers. Delayed-type from an immunological standpoint is collagen. Type hypersensitivity (DTH) is also known as Type IV II collagen is known for its ability to induce hypersensitivity under the Gell and Coombs autoimmune arthritis [23], and, while types I and III classification and by the general population as do not initiate this response, research has indicated contact dermatitis. The DTH is mediated by antigen- that each of these collagen types is immunogenic presenting cells and T-cells, specifically the CD-4+ [24]. The immune responses to collagen types I, II, subset of the T-cell popoulation. Since the immune and III in a rabbit experimental model was reported system is multifaceted, the question arises as to how to be T cell-mediated with minimal humoral response exposure to a biomaterial affects the various [25]. Others have indicated that T cells play a pivotal components of the immune system. There will role in macrophage recruitment and giant cell undoubtedly be acute inflammation, resulting from formation in reaction to cross-linked dermal sheep the cut of a scalpel that compromises the integrity of collagen [26], suggesting that inflammation in the skin, a major component of innate immunity. response to collagen may be modulated by Furthermore, should the biomaterial suppress or controlling T cell activation. In addition, these stimulate the immune system inappropriately, such researchers demonstrated that TNF-α and IFN-γ were modulation could be detrimental to the integrity of not responsible for the onset of the foreign-body the biomaterial as well as the immunocompetance of reaction to collagen in mice [27, 28]. Recently, it was reported that collagen fragments alone did not With regard to biomaterial effects on innate characterization of the immune response to these immunity, the primary focus has been on the activity collagen fragments, using LPS-activated monocytes, of phagocytic cells. The widely used synthetic demonstrated that IL-1 levels increased or decreased, vascular prosthetics made of Dacron and expanded depending on the peptide sequences present in the polytetrafluoroethelyne (e-PTFE) have been shown to fragments. However, it is impossible to understand result in the reduction of phagocyte concentration in the significance and implications of these results human donor blood [13]. It has also been well without first having some idea as to whether they documented that e-PTFE and other biomedical result in biologically relevant effects on the immune polymers activate macrophages and increase system. A more complete evaluation of the effects production of both IL-1 and TNF-α [14-18]. on the various components of the immune system is Recently, Brodbeck et al. have demonstrated that the needed, even for this extensively studied biomaterial. Journal of Engineered Fibers and Fabrics One polymer currently receiving attention is ranging from 6.0 μm to 15.9 μm. Missing from this polydioxanone (PDO), a bioresorbable polymer that puzzle, however, is a determination as to whether is completely degraded in vivo within six months. smaller diameter fibrous biomaterials generate PDO vascular prostheses have been shown to be less different immune responses. Our research has thrombogenic than both PGA and Dacron synthetic suggested that the biocompatibility of a material grafts [30], thus making them desirable to use in changes significantly between the micron and the vascular tissue engineering applications. However, nanometer fiber diameter levels [37], a suggestion like with other synthetic polymers, the use of PDO that is not difficult to believe given the results alone does not promote cell infiltration in vivo [31]. reported by Sanders et al. This further emphasizes It has been reported that PDO suture (PDSTM-II) the need for an examination of the immune response produced no sign of acute or chronic toxicological to electrospun sub-micron fibrous biomaterials. effects in any tissues or organs following a six-month in vivo implantation [32], indicating that the long- So, if study of the immune responses to biomaterials term effects of this polymer may be negligible. is truly critical, why is there so little organized focus However, studies at time points less than 6 months, on such research today? Immune cell interaction during which the polymer is degrading, may indicate with a biomaterial following implantation is effects on the immune system, thus earlier time unavoidable. Even when bioresorbable materials are points must be evaluated in order to determine the used (as temporary scaffolds encouraging formation specific effects of PDO prior to complete resorption of native tissue), cells of the immune system have of the polymer. In contrast, PDSTM-II suture has ample opportunity for interaction with these been shown to induce fibrosis after 14 days in a rat materials; interaction times with permanent materials subcutaneous implantation model [33]. Others have are likely not much longer, as the formation of a demonstrated that soluble suture fragments of PDO fibrotic capsule around permanent materials is had minimal effects on the macrophage as compared probable, depending on the nature of the biomaterial. to silk, nylon, and polyglactin [34]. Each of the suture materials examined therein were indicated to Nearly all published research on immune responses release “immunotoxic factors,” although no specific to biomaterials has presented too narrow a focus, a factors were identified, nor were any cell types other view too mechanistic with little characterization of than the macrophage examined. PDO is believed to the overall immune response first. The result is a be less immunogenic than PGA and PLA [35], yet breadth of knowledge that has no contextual this too remains to be examined thoroughly. Again, foundation. With the appropriate biological assays as it is imperative that an overall picture of immune tools, a more complete picture of immunomodulation responses be obtained prior to assertions regarding a resulting from exposure to biomaterials can be biomaterial’s relative effects on the immune system. achieved than is possible from the current approaches. An assortment of assays described by Work in our lab focuses on the design and evaluation Luster et al. [48] which are typically used in of tissue engineering scaffolds fabricated by immunotoxicological evaluations of pharmacological electrospinning. We have successfully electrospun a compounds can provide a more complete appraisal of variety of synthetic polymers [35-38], natural acquired and innate immunity than is currently being polymers [39-43], and blends of the two [31, 44, 45] achieved with the limited assays currently in use by for use in a variety of tissue engineering applications. With the sudden increased interest of biomedical engineers in electrospinning, it is important that the An initial approach for examining immune responses effects of electrospun biomaterials upon the immune following in vitro exposure of a biomaterial could system be examined. However, only limited consist of evaluating effects on: cell-mediated published research has examined the immune immunity by examining T cell proliferation alone or in the presence of anti-CD3 antibody or the T-cell mitogen ConA [49], the humoral immune response by assessing effects on B cell proliferation (LPS- Sanders, et al. have published regarding the influence stimulated or F(ab’)2 and IL-4 – stimulated [49]) and of polymer fiber diameter on fibrous capsule antibody production (Mishell-Dutton hemolytic AFC formation and thickness [46, 47], demonstrating that assay [50]), and innate responses by examining implantation of smaller diameter fibers (1.0 μm to 5.9 chemotaxis [51] and activity [52] of phagocytic cells, μm) resulted in thinner or no fibrotic capsule in addition to NK cell activity [53, 54]. In each of formation when compared to fibers with diameters these assays, splenocyte exposure to the biomaterial

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