Assays on the Influence of Biomaterials on Allogeneic Rejection in Tissue Engineering

Abstract

In tissue engineering, innate responses to biomaterial scaffolds will affect rejection of allogeneic cells. Biomaterials directly influence innate and adaptive immune cell adhesion, reactive oxygen intermediate production, cytokine secretion, nuclear factor-kappa B nuclear translocation, gene expression, and cell surface markers, all of which are likely to affect allogeneic rejection responses. A major goal in tissue engineering is to induce transplant tolerance, potentially by manipulating the biomaterial component. This review describes methods of measuring responses of macrophages, dendritic cells, and T cells stimulated in vitro and in vivo and addresses key factors in assay development. Such tests include mixed leukocyte reactions, enzyme-linked immunosorbent spot assays, trans-vivo delayed-type hypersensitivity assays, and measurement of dendritic cell subsets and anti-donor antibodies; we propose extending these studies to tissue engineering.

Introduction

Although there are many possible permutations, most proposed tissue engineered constructs have two components: a biomaterial scaffold and allogeneic cells. As such, the function of the final construct depends on the recipient response to the combination of biomaterial and cells. There are assays established that test the response to biomaterials alone and to allogeneic transplants alone but few that have been used to test the hypothesis that biomaterials will affect allogeneic rejection. In considering immune responses to tissue-engineered constructs, there are two basic goals: minimizing the immune response to the construct using thoughtful design and understanding the response well enough that anti-rejection strategies can be designed that are effective and have few side effects. Ideally, cell and biomaterial selection (the focus of this review) will induce specific transplant tolerance, or at least minimize rejection.

To facilitate efficient biomaterial selection, quantitative in vitro assays are an important tool. In some cases, more information is obtained about the complex interactions of the immune rejection response through in vivo implantation followed by in vitro assays. This review will address questions of how to set up such experiments and how to measure the outcomes.

In Vitro Assays

Setting up meaningful in vitro assays

To simulate in vivo implantation of a tissue-engineered construct, it is critical that proper attention be paid to the delivery of the biomaterial in vitro and to the choice of relevant responder cells. Most tissue-engineered constructs include differentiated cells seeded on a large, three-dimensional biomaterial scaffold to which many different cells involved in wound healing and the immune system respond. Determining the critical components of an in vitro experiment requires careful consideration. Methods to analyze the effect of in vitro contact between materials and cells are summarized in Table 1.

Table 1.

Methods of Analyzing in Vitro Exposure of Cells to Biomaterials

Measurement	Interpretation
Biomaterials + innate cells
Reactive oxygen intermediates	Local damage leading to damage-associated molecular-pattern molecules
Enzymes	Local damage leading to damage-associated molecular-pattern molecules
Cytokines	Inflammatory signalling
Cell surface markers	Costimulation
Receptor blockade	Mechanistic studies
Biomaterials + adaptive cells
Cytokines	T cell subset (e.g., Th1, Th2, Th17, Tr1)
Cell surface markers	Activation
Proliferation	Activation
Biomaterials + innate cells + adaptive cells
Proliferation	Rejection vs. suppression
Cell surface markers	Activation
Cytokines	T cell subset (e.g., Th1, Th2, Th17, Tr1)
Blockades (e.g., CD4+ or receptor)	Mechanistic studies
Differentiation	Activation

Biomaterial mode of interaction in vitro

It is impossible to reproduce the dynamic in vivo environment precisely. If one were to create a complete construct, it would be a challenge to expose cells of the innate or adaptive immune system to it in vitro; in static culture, these cells would tend to settle beneath the construct and not contact it, as would occur in vivo. Perfusion of the construct with responder cells might be an appropriate alternative, but bringing this additional technical complexity to the system precludes rapid screening of appropriate biomaterials. The benefits of an in vitro system ought to include simplicity, reproducibility, simple interpretation, and low cost. As such, we must consider exposure of responder cells to biomaterials in static culture.

One common approach is simply to coat a tissue culture flask or plate with the biomaterial(s) of choice, usually using solvent casting (e.g.,^1–3). Responder cells would then be seeded on the film, ensuring contact. The cells would be unable to engulf it, which would mirror “frustrated phagocytosis” of relatively large scaffolds. On the other hand, in cases where cell adhesion influences survival (or even response) in culture, this might produce artefacts. In vivo, if a material does not induce cell adhesion, brief contact with responder cells will ensue, which might or might not lead to activation. In contrast, in vitro culture on a film causes prolonged contact with the biomaterial, potentially leading to a dramatically different outcome. Indeed, it has been shown that adhesion of macrophages is negatively correlated with inflammatory cytokine production,⁴ which might be due to adherence-triggered apoptosis. Similarly, polyethylene glycol-grafted non-adherent surfaces have been shown to activate macrophages,⁵ whereas arginine-glycine-aspartate–coated adherent surfaces cause macrophage fusion.⁶

In testing blood compatibility, Sefton et al. used a method that had nearly the ease of static culture combined with the significance of low shear contact.^7–9 They incubated blood cells in biomaterial tubes on a rocker platform. In such a set-up, cell adhesion plays a smaller role, and the consequences of flow are simulated.

If biomaterials interact directly with cellular receptors, a straightforward way of testing would be to deliver the biomaterial in a soluble form. This is not possible in cases in which the biomaterial solvent is not compatible with cell culture, but for some cross-linked materials, the non-cross-linked polymer becomes water soluble. In these convenient cases, tests can proceed much as any test of soluble ligands. For example, macrophage responses to soluble alginate^10–13 and chitosan^14,15 have been tested. However, we have shown with alginate that cross-linking decreases macrophage responses (unpublished data).

Another common way of exposing responder cells to biomaterials is to deliver microparticles or microspheres. Here, the responder cells are established in culture, and microspheres are subsequently added to the medium. This method has the benefit of delivering the material with minimum disruption to the cells. There is little influence of adhesion to the test material, and the cells are in stable culture, minimizing the variability of the treatment. Depending on the size and nature of the microspheres and the type of responder cell, the particles will often be taken into the cell by phagocytosis, endocytosis, or pinocytosis.^16–18

Microparticle delivery is commonly studied in the vaccine adjuvant literature, in which poly(lactic-co-glycolic acid) (PLGA) microspheres have been proposed as vaccine delivery vehicles. Although there is a great deal that tissue engineers could learn from biomaterial adjuvant research, there are factors that must be considered in using microspheres in vitro to probe the potential effects of biomaterial scaffolds on immune responses in tissue engineering. Typically, a scaffold is too large for the cells to take up, whereas microspheres are often successfully phagocytosed. Because little is understood about the way in which cells respond to biomaterials, it is difficult to predict the influence of the physical form of the biomaterial. It is possible that phagocytosable particles and non-phagocytosable scaffolds will have different effects on cell activation. Non-phagocytosable materials have the capacity to cause persistent stimulation of cells, whereas it is possible that biomaterials taken up into the cell lose their activating power. For example, it has been demonstrated that PLGA scaffolds induced higher antibody titers after implantation than do PLGA microcapsules, although this could be attributed to altered levels of concomitant tissue damage.¹⁹ It has also been demonstrated that micro- and nano-scale structures differentially alter responses of smooth muscle cells,²⁰ macrophages,^21,22 and soft tissue.²³ As a preliminary screen, however, using microspheres has considerable advantages in culture. To determine effects of specific constructs, it would be advisable to test them in their final form but as a subsequent assay.

Responder cell selection for in vitro assays

No matter the responder cells selected, a problem with in vitro tests is the response to the culture and isolation materials. Just as construct biomaterials have the potential to activate cells, so too do tissue culture polystyrene, centrifuge tubes or syringes. Tissue culture polystyrene has been shown to activate U937 cells.²⁴ Thus, care must be taken to use appropriate controls.

Cell lines or primary cells can be used to test in vitro responses to biomaterials. Primary cells can be used directly in mixed culture, sorted into pure culture, or differentiated before use. Cell lines are more appealing than primary cells, because results are stable and reproducible, culture is relatively straightforward, and they are relatively inexpensive. One drawback is that immortalized cell lines sometimes display different responses than primary cells.²⁵ In addition, in immunology, a complex mix of cell types often interacts in responding to a challenge. This is difficult to reproduce using cell lines.

Depending on the assay, there are many different cell lines available that are relevant for testing responses to biomaterials in vitro. Implanted biomaterials may affect many different cell types, directly or indirectly. Anderson has described the classic sequence of response.²⁶ First to respond to biomaterial implantation are neutrophils. The neutrophil response has been studied but not in the same depth as the macrophage response, in part because it has been shown that the effect on neutrophils is primarily due to wounding rather than to the biomaterials themselves; sham surgeries elicit the same neutrophil response as biomaterial implantations.²⁷

From histological studies, macrophages are usually the second responders to biomaterial implantation. There are many macrophage and monocyte cell lines, mouse and human, available. The purpose of this review is not to provide an exhaustive list of cells and protocols but merely to draw attention to relevant variables in setting up in assays.

In the field of device implantation, there has been little investigation of the role of dendritic cells, because histologically, they have rarely been identified near implants. However, if these professional antigen-presenting cells (APCs) interact with biomaterials, their role in altering adaptive immunity will be important. There are a small number of dendritic cell lines available.

It is common to observe lymphocytes in the vicinity of a biomaterial implant, but little is known about direct interactions with biomaterials; it is usually (but not always) assumed that there are none. Although there are T cell and B cell lines, they have some significant differences from primary cells that mean they are not relevant for all assays.

Primary cells offer their own set of advantages and limitations. For understanding innate responses to the biomaterial component alone, there are several methods of cell isolation and differentiation for macrophages and dendritic cells. An excellent resource for detailed isolation protocols is Current Protocols in Immunology.²⁸ For human systems, the most accessible source of cells is from blood. Monocytes derived from peripheral blood mononuclear cells (PBMCs) can be differentiated into macrophages or dendritic cells. Because these cells cannot be harvested and isolated without some exposure to biomaterials and because differentiation is required, effects of in vitro biomaterial stimulation might be masked. In addition, there is accumulating evidence that the tissue source of dendritic cells will alter their reactivity. Still, peripheral blood-derived macrophages and dendritic cells give access to a human-based system that has clinical relevance. Blood-derived white blood cells (leukocytes) also contain lymphocytes, thus allowing assays of the adaptive immune system such as the mixed leukocyte reaction (MLR).

A wider range of cell sources is available from animal models. The focus will be on mice because of the range of reagents and knockout models available. In addition to blood-derived cells, macrophages are also often derived from the peritoneal cavity, bone marrow, and the spleen. These cells have a range of maturation phenotypes upon extraction and might need further differentiation. Because dendritic cells are low in number and resident in tissues, they are rarely extracted as mature cells; usually, monocytes are exposed to maturation stimuli. For adaptive immune studies (e.g. MLRs), splenocytes or cells from peripheral lymph nodes are usually used. Human and mouse cells can be fractionated using a variety of techniques to parse the effects of biomaterials.

In cases in which adaptive responses to allogeneically transplanted cells are to be measured, attention must be paid to the stimulator and responder cells. The degree of matching of the major histocompatibility complexes (MHCs) or human leukocyte antigens (HLAs) between the cells will affect the level of response. Ideally, one would assay the worst possible scenario, immunologically. In addition, not all stimulator cells (i.e., the cells representing those of the tissue-engineered construct in an in vitro assay) have the same level of MHC/HLA expression. Undifferentiated human mesenchymal stem cells, for example, express low levels of HLA and show immunomodulatory properties.^29,30 Of interest to tissue engineers, there is some evidence that cells differentiated from stem cells maintain their immunomodulatory activity,³¹ although this is controversial. Even genetically matched stem cells will potentially cause natural killer cell responses if MHC I expression is low. An excellent review of the potential of stem cells from a transplant perspective has recently been published.³² It is thus important to use cells that are reflective of the actual cells to be used in the tissue-engineered construct.

Measuring the direct effect of the biomaterial on innate immune cells

The primary effect of the biomaterial component is likely to be on cells of the innate immune system, such as macrophages and dendritic cells. There are a number of ways of measuring the level of activation for biomaterial screening purposes. Some assays to elucidate the mechanism by which biomaterials are recognized will also be described.

Viability and adhesion of macrophages seeded on biomaterial surfaces is sometimes used as a measure of “biocompatibility.” As noted above, the significance of in vitro adhesion on in vivo responses is not altogether clear. Ainslie³³ used cell survival and adhesion of macrophages on polytetrafluoroethylene nanofibrous surfaces as such an indicator. Adhesion to surfaces is linked to macrophage fusion and subsequent foreign body giant cell formation,³⁴ which is a concern for regeneration but not for subsequent influences on adaptive immunity. Biomaterials have been shown to induce other activation markers that are important in device design but less likely to influence immune responses. For example, activated U937 cells release degradative enzymes including esterase and acid phosphatase.³⁵

One simple and commonly used assay measures the release of cytokines by cells incubated with biomaterials. The supernatant is removed at times varying between 12 and 96 h, and the cytokine levels are quantified using enzyme-linked immunosorbent assays (ELISAs). In some cases, intracellular cytokine levels are measured using flow cytometry, or messenger RNA (mRNA) levels are measured using polymerase chain reaction. Cytokines will affect inflammation and will also influence activation levels of lymphocytes. This will potentially compromise the function of the tissue-engineered construct and induce further “danger signals”³⁶ that will stimulate innate immune responses.

Production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-1 beta (IL-1β) is most often monitored. For example, rat alveolar macrophages upregulate TNF-α and IL-6 mRNA in response to a range of biomaterials.³⁷ Similarly, silicon dioxide nanoparticles induced TNF-α and IL-1β.³⁸ These three cytokines are a small subset of a much wider range of cytokines that have been analyzed. For example, RAW 264.7 cells were shown to release high levels of TNF-α, granulocyte colony-stimulating factor (CSF), monocyte chemoattractant protein-1, regulated upon activation normal T cell expressed and secreted, granulocyte macrophage CSF, and eotaxin in response to alginate oligosaccharides.¹² Cytokines promoting Th2 bias, IL-13 and IL-25 were shown to be upregulated in alveolar macrophages in response titanium dioxide particles.³⁹ Hydroxyapatite particles upregulated IL-18 in human monocytes.⁴⁰ Other cytokines including IL-1α and IL-9,¹² interferon-gamma, IL-4, and IL-5³³ have been studied but have not been shown to be strongly upregulated by biomaterials.

Flow cytometry of cells exposed to biomaterials displays changes in surface markers. Babensee et al. have shown alterations particularly in the costimulatory molecules CD80 and CD86 and maturation marker CD83 in human and mouse dendritic cells due to biomaterial contact.^41–43 They also studied CD40 and MHC expression, but these were not significantly altered in their experimental system. Such changes would have important consequences for efficiency of antigen presentation to T lymphocytes. Differentiation of dendritic cells has the potential to affect the immunological outcome profoundly; antigen presentation through immature dendritic cells has been associated with production of regulatory T cells.^44,45 Similarly, plasmacytoid dendritic cells have been associated with allotolerance,⁴⁶ whereas myeloid dendritic cells have been used in immunotherapy against cancer.⁴⁷ Thus, the dendritic cell phenotype induced by biomaterial contact has the potential to cause accelerated rejection or immune tolerance.

Macrophages and neutrophils will often release reactive oxygen intermediates (ROIs) as a result of inflammatory stimulation. Increased levels of ROI release have been measured in response to biomaterial stimulation. Yamamoto employed a superoxide-reactive chemiluminescence producer and an optical fiber probe to demonstrate ROI production in response to blood dialysis membranes.⁴⁸ Similarly, macrophages stimulated in vitro with PLGA microspheres showed increased ROI production.⁴⁹ Among other possible reactions, increased ROI levels will induce local tissue damage that can compromise the function of the cells in the tissue-engineered construct, which would cause subsequent innate-activating danger signals — an inflammatory feedback loop.

The effect of biomaterials on gene expression in innate immune cells has also been studied. The advent of microarray technology means that it is now possible to study a large number of responses simultaneously. There are “inflammatory” microarrays available that narrow the spectrum of targets. Such systems are sensitive to variability in experimental conditions, but with careful selection of controls, in vitro biomaterial testing with microarrays has great promise. Some preliminary work has shown the efficacy of microarrays in highlighting responses to particles.⁵⁰

In addition to screening the effects of biomaterials on innate immune cells, determining the mechanism of activation is important to design improved biomaterials. It has been hypothesized that some polymeric biomaterials signal through Toll-like receptors (TLRs) on innate immune cells.¹⁴ Metallic and ceramic nanoparticles do cause upregulation of TLR3, TLR7, and TLR9.³⁸ One key downstream effect of signalling though many of the TLRs is translocation of the transcription factor nuclear factor-kappa B (NF-κB) from the cytoplasm to the nucleus.⁵¹ This can be monitored using immunofluorescence or by separating nuclear and cytoplasmic fractions and running Western blots or ELISAs.⁴¹ Electrophoretic mobility shift assays also show if NF-κB has bound to DNA,⁵² and there are antibodies that distinguish the active and inactive forms of NF-κB.⁵³ On its own, NF-κB analysis can be used as a screening tool, but it can also be powerfully combined with the use of cells from receptor knockout sources. It seems that receptors are involved in biomaterial recognition.⁵⁴ Recently, there has been interesting work showing that antibodies to TLR2 and TLR4 block RAW 264.7 responses to alginate oligosaccharides¹¹ and that antibodies to TLR4, CD14, and CD3 block the effect of chitooligosaccharides.¹⁴ Similarly, the inflammatory effect of hydroxyapatite disappears in TLR4 −/− macrophages.⁵³ Scavenger receptors have also been implicated; the lung inflammatory defense against particles was lower in mice that were −/− for the macrophage receptor with collagenous structure (MARCO)⁵⁵ and for SR-AI/II −/− mice.⁵⁶

Measuring the direct effect of the biomaterial on adaptive immune cells

It is generally assumed that biomaterials do not interact directly with cells of the adaptive immune system (e.g., T and B lymphocytes), despite the observation that lymphocytes are attracted to biomaterial implant sites.⁵⁷ Most biomaterials are unlikely to elicit an antigen-specific response directly in T cells; they do not have MHC molecules, and most biomaterials are not protein based. Notable exceptions are emerging as protein–peptide engineering becomes more developed. Biomaterials based on collagen, elastin, and spider silk (to name just a few) have been proposed.^58–62 Even protein-like biomaterials based on engineered amino acids have been designed.⁶³ These materials have the potential to activate adaptive immune responses. Additionally, some biomaterials extracted from xenogeneic sources have been used. A biomaterial based on porcine small intestinal submucosa has been shown to cause antibody production, although with no clinically adverse consequences.⁶⁴ Nonetheless, most biomaterials proposed for tissue engineering are polymeric and not polypeptides.

B cells can recognize a wider range of antigens (including polysaccharides) than T cells, leaving open the possibility that B cells will produce antibodies specific to biomaterials. It is also well known that proteins adhere strongly and rapidly to most polymeric biomaterials.⁶⁵ Particularly during the breast implant controversy, many researchers proposed that native proteins adhered to biomaterials became rearranged, exposing new epitopes⁶⁶ that were recognized as antigens, leading to autoimmunity. The lymphocyte activation by such cryptic epitopes is possible but has not been conclusively proven.

Stimulation of lymphocytes could occur through a number of mechanisms. The material itself could stimulate the cells by unknown means. Alternately, proteins bound to the biomaterial surface (e.g., opsonins such as non-specific antibodies that activate innate cells) might cause activation. Some proteins change conformation and become activated as a result of biomaterial contact. For example, biomaterials have been shown to cause formation of complement fragments;^67,68 in turn, such complement fragments have been shown to activate leukocytes⁶⁹ and even induce regulatory T cells.⁷⁰ If biomaterials directly activate lymphocytes, this will have important consequences for the reaction to transplanted allogeneic cells. Recent evidence has emerged that T cells also express TLRs⁷¹ and that ligation of the TLRs on CD4+ cells is involved in regulating regulatory T cells.⁷²

The most straightforward way to monitor the effect of biomaterials on lymphocytes is simply to co-incubate the biomaterials in vitro with purified lymphocytes. Measuring proliferation, usually through the incorporation of tritiated thymidine or bromodeoxyuridine into the DNA of dividing cells monitors lymphocyte response. Otsuka recently showed that phenylboronic acid residues and surface wettability of polymers affected lymphocyte proliferation in vitro.⁷³

For T cells in particular, division does not necessarily indicate function. The cytokines they produce influence the outcome. Th1 cells (with signature interferon-gamma (IFN-γ) secretion) are typically associated with rejection, whereas Th2 cells (with archetypal IL-4 production) are controversially linked to a less robust rejection response.^74,75 Newly identified Th17 cells (with IL-17 secretion) are associated with autoimmunity and might be involved in transplant rejection.⁷⁶ Finally, a range of regulatory T cells (e.g., Foxp+CD4+CD25+ cells, IL-10–secreting Tr1 cells, and transforming growth factor beta (TGF-β) secreting Th3 cells) have been associated with transplant tolerance.^77,78 Tolerance induction is clearly desirable in tissue engineering. Thus it would be informative to measure the cytokines secreted into the supernatant of these cultures. Individual ELISA kits could be used; there are also multiplex beads that measure a panel of T helper cytokines using flow cytometry.

With a slightly different perspective, one interesting study was done that examined the effects of biomaterials on cell differentiation during primary bone marrow cell culture. When a tissue-engineered construct is implanted, it causes wounding, which has in turn been shown to cause recruitment of bone marrow cells.^79,80 When bone marrow cells were cultured with polyethylene or cobalt chromium molybdenum particles, they induced proliferation of CD14+ (monocyte/macrophage) and CD66b (granulocyte) cells, respectively.⁸¹ Markers for T cells (CD4+), B cells (CD19+), and hematopoietic stem cells (CD34+) were also analyzed.

Measuring the overall effect of the biomaterial on adaptive immune cells

Because biomaterials cause inflammation in vivo, thus activating macrophages (APCs), most researchers in the field believe that influences of biomaterials on adaptive responses will occur indirectly because of activation of APCs. However, simply knowing the level to which a given biomaterial activates an APC in vitro will not predict the effect on the adaptive immune response. Thus it is necessary to measure the influence of biomaterials in an assay more representative of the rejection response.

Biomaterials incubated with a mixture of cells will show the overall response of innate and adaptive immunity, although it will not give information directly about the effect of biomaterials on rejection. Borges et al. incubated unfractionated splenocytes with soluble chitosan and alginate.⁸² They found that an early cell activation antigen, CD69, was upregulated on B cells and T cells, although proliferation was unaffected. It is not clear whether interactions between innate and adaptive cells influenced the result.

Anderson et al. have shown that interactions between ymphocytes and monocytes influence the response to biomaterials, even in the absence of antigen. Lymphocytes incubated with tissue culture polystyrene and poly(ethylene terephthalate)–based biomaterials increased monocyte attachment, and monocytes incubated with biomaterials increased lymphocyte proliferation in a material-chemistry-dependent manner.^83,84 Direct contact was not required. Such studies illustrate the complex interactions that occur in the foreign body response.

The MLR is the standard in vitro method in transplantation to test for allogeneic rejection. Typically, the stimulator cells are treated using gamma irradiation or mitomycin C to prevent proliferation.²⁸ For testing tissue-engineered constructs, the stimulator cells will be the cells that would be included in the construct. Responder cells (splenocytes in murine models; PBMCs in human models) are incubated with the stimulator cells, usually for 4 to 5 days, and the resulting proliferation is measured. MLRs are effective tools for measuring allogeneic responses, because up to 10% of T cells will respond directly to MHC mismatches. This means that proliferation in response to primary stimulation is measurable. This technique is not, however, sensitive to primary stimulation with processed antigens presented by recipient APCs (indirect presentation), although it can measure memory responses to indirect stimulation. Indeed, the MLR has been shown not to reflect clinical tolerance.^85,86 This restricts the ways in which this assay can be used in vitro without in vivo priming in measuring indirect effects of biomaterial stimulation.

One can simply add the biomaterials to an MLR and determine the effect on proliferation. Here, it is important to include appropriate controls to determine the effect of the material in the absence of allogeneic stimulation. Babensee et al. has shown that dendritic cells pre-incubated with PLGA and, to a lesser extent, agarose stimulate greater allogeneic T cell proliferation.^41,43 Although an excellent indication of dendritic cell activation, this test reflects direct rather than indirect rejection responses. Depletion of cells from these assays (e.g., by depleting antibodies) will determine the affected cells, and assaying the cytokines in the supernatant will give more information about the type of response (e.g., Th1, Th2, or Tr1).

In vaccine adjuvant research, tools have been developed to further parse responses, even without primary stimulation in vivo. For a number of model protein antigens, researchers have determined the dominant peptides that are present in the MHCI and MHCII binding clefts. Antibodies have been developed that recognize the peptides in the binding cleft. Similarly, many tetramers are commercially available that bind to specific T cell receptors, usually on CD8+ cells. Moreover, T cell receptor transgenic mice have been developed in which all the T cells respond to a specific peptide. For example, OT-I mice are transgenic for the CD8+ T cell receptor, recognizing peptides 257-264 of hen egg ovalbumin (OVA) in the context of H-2 Kb. Also, OT-II transgenic mice express the CD4+ T cell receptor specific for OVA323–339. Because every T cell is responsive, primary MLRs become possible. Using such systems, Hamdy et al. have demonstrated that recipient dendritic cells efficiently process OVA delivered with PLGA nanoparticles and present them to recipient T cells.⁸⁷ In allogeneic transplantation models, similar experimental tools are not yet available. The vaccine adjuvant work suggests that biomaterials are likely to influence the indirect component of the allogeneic rejection response.

In vivo assays

Setting up in vivo assays

In striving to understand innate and adaptive responses to tissue-engineered constructs, in vivo models provide information about complex interactions that are impossible to capture using in vitro assays. The most-accurate outcomes would be measured by testing a final “perfected” version of a tissue-engineered construct in the target location, but to incorporate feedback from immune responses, it is beneficial to create simpler models that provide information about appropriate biomaterial selection before full construct development. There are several key variables to consider in designing in vivo models. First, the model construct geometry should be reflective of the final design. As discussed earlier, the size of the biomaterial features relative to that of the cells is likely to influence the response. Second, the implant location will probably affect the immune respons.⁸⁸ Immune-privileged sites such as the cornea or testis will give dramatically different responses,^89,90 but more-subtle location choices such as intraperitoneal versus subcutaneous implantation might also alter responses. Third, as with in vitro assays, the selection of cells should reflect the “worst case” immunologically. For example, if a range of cells will be included in a proposed construct, the ones that have the highest MHC expression should be used. In addition, ensuring MHC mismatch between donor cells and recipient is important. Finally, surgical technique is critical; the greater the level of wounding, the higher the “danger signals” to the innate immune system.³⁶ Careful attention to controls should include naïve animals, sham surgeries, cells alone, and biomaterials alone. Methods to analyze the effect of in vivo implantation of constructs with biomaterials and cells are summarized in Table 2.

Table 2.

Methods of Analyzing Immune Responses to Implanted Cell–Biomaterial Constructs

Measurement	Interpretation
Local responses
Transplanted cell survival	Function
Histology	Inflammatory or rejection response
Cytokines	T cell subset (e.g. Th1, Th2, Th17, Tr1); inflammation
Local cell activation (e.g., from lavage)	Inflammatory or rejection responses
In situ inflammation markers	Local damage
Systemic responses
Anti-donor human leukocyte antigen or major histocompatibility complex antibodies in blood	Tolerance vs. rejection
PDC:mDC ratio in blood	Tolerance vs. rejection
Indirectly measured immune responses
Recipient splenocyte rechallenge with donor cells vs. third-party splenocytes (proliferation; cytokine secretion)	Hyporesponsiveness = less rejection
Enzyme-linked immunosorbent spot assays (rechallenge as above)	Memory vs. primary responses; T cell subset activation
Trans-vivo delayed-type hypersensitivity	Tolerance vs. rejection (also mechanistic)

Measuring local outcomes

Straight monitoring of the local environment around an implant is informative, although the mechanism is not elucidated, and survival of transplanted cells is an essential indicator. These methods are, however, often too crude to distinguish subtle (but ultimately important) responses to different biomaterials. This review is primarily intended to cover in vitro analysis of responses (with or without implantation) and so will examine local responses and transplant survival in only a cursory manner.

Perpetual survival of the cells in a tissue-engineered construct is the desired outcome. It is a reasonable assumption that, without using anti-rejection drugs, allogeneic cells will not survive in vivo, but real-time monitoring of in vivo cell survival is not easy. There are some models in which the cells perform a function that can easily be monitored, as with islet transplants altering blood glucose levels (e.g., ^91,92). For many models, though, the only method of monitoring function is to sacrifice the animal and look for markers that distinguish donor cells from recipient cells within the construct. Ideally, cell markers (metabolic or visual) will be introduced that allow real-time monitoring of the viability of implanted cells, without sacrificing the animal. Noninvasive monitoring has been accomplished using proton NMR imaged choline production from betaTC3 cells.⁹³ Magnetic resonance imaging has also been used to followed cells in vivo, where the cells have been labeled with superparamagnetic iron oxide.⁹⁴ One intriguing study used cells that were transfected such that NF-κB activation caused EGFP production and fluorescence, thus allowing non-invasive monitoring of stress to cells within an implanted construct.⁹⁵

Histology of explanted tissue-engineered constructs will provide valuable information about cell survival and about recipient immune cells active at the implantation site. If the construct were implanted in the peritoneal cavity, local cells could be harvested using peritoneal lavage. The cells in the lavage can then be analyzed after a cytospin and H&E stain or analyzed using a flow cytometer after the cells are labeled with antibodies. Recipient cells attracted to the implant site can also be analyzed for gene expression profile, including using microarrays.

Ho et al. developed one elegant technique for visualizing responses to implants.⁹⁶ Briefly, they created a transgenic mouse that had a luciferase reporter gene linked to a NF-κB-responsive promoter. Thus, when NF-κB was stimulated to translocate to the nucleus, the cells emitted light that was monitored using in vivo bioluminescent imaging, allowing ongoing, non-invasive observation of the inflammatory response.

Measuring the effect on adaptive immunity

Evidence is accumulating that biomaterials affect innate immune responses. In turn, it has been hypothesized that adaptive responses will be altered. A challenge in developing assays to test this in an allogeneic setting is that biomaterials will thus affect the indirect component of the rejection response. Although indirect responses (recipient APCs activating recipient T cells) are sufficient to cause rejection,^97,98 the robust primary direct response (recipient CD8+ T cells interacting directly with donor cells) that dominates allogeneic transplantation masks them in many assays. Tolerance responses are primarily induced through indirect activation. One tantalizing possibility in tissue engineering is that, through appropriate biomaterial and cell selection, a tolerant state could be induced, negating the need for a lifetime of anti-rejection drugs. To measure the influence of biomaterials on the indirect response, the construct is implanted (priming the immune system), and subsequent assays are performed. These include assays that examine the effect of T cells, B cells, and dendritic cells.

As described above, the MLR is the classic assay in allogeneic transplantation. When determining the level of primary activation in vivo, recipient splenocytes are re-challenged with donor cells or with allogeneic third-party cells. The secondary response to the donor cells in rejecting recipients is greater than the primary response to the third-party cells.^99,100 Hyporesponsiveness in this assay is correlated with less-robust rejection (e.g., effective immunosuppression) but has not been linked to specific tolerance.⁸⁵ The biomaterial component of the construct will potentially alter the magnitude of this response. Indeed, a similar assay has been performed in a xenogeneic model, in which indirect rejection dominates the T cell component. There, a biomaterial combination of agarose and a copolymer of hydroxymethylmethacrylate and methylmethacrylate implanted coincident with a xenogeneic skin graft was shown to transiently suppress the second-set MLR proliferative response.¹⁰¹ This demonstrated that biomaterials are not necessarily activating and deleterious in tissue engineering; it is likely that they will alter immune responses but not necessarily for the worse. In an allogeneic system, the MLR does not distinguish between direct and indirect responses. Direct and primary responses will mask the influence that biomaterials have on the indirect and memory response.¹⁰²

The enzyme-linked immunosorbent spot (ELISPOT) assay overcomes some of the limitations of the MLR in these circumstances. Responder cells from in vivo primed animals are challenged with stimulator cells at a lower concentration than in the MLR. The reaction occurs over a much shorter time than the MLR (24 h) on a cytokine-coated plate. After incubation, the plate is developed such that each cell that produced the target cytokine causes a spot. Because of the short incubation time, memory responses are more easily distinguished from primary responses. In addition, the T cell subsets elicited using transplantation can be distinguished by using ELISPOTS for cytokines produced by Th1 cells (e.g., IFN-γ, IL-2), Th2 cells (e.g., IL-4, IL-5), or even Tr1 cells (IL-10), although this assay does not work to detect TGF-β-producing Th3 cells.¹⁰²

The trans-vivo delayed-type hypersensitivity assay highlights the indirect and regulatory components of transplant responses. Developed by VanBuskirk et al. to detect tolerance in human transplant recipients, it takes advantage of memory CD4+-dependent inflammation.^103–107 Recipient PBMCs are mixed with freeze-thawed donor cell lysate and injected into the footpad of a severe combined immunodeficiency mouse. Samples from donor-sensitized patients led to footpad swelling within 24 h. Non-swelling regulatory responses could be distinguished from non-regulatory responses by including a recall antigen; regulatory cells suppressed the swelling response to the recall antigen. Furthermore, by using antibodies to IL-10 and TGF-β, the type of regulatory cell could be determined.

As discussed above, plasmacytoid dendritic cells have been implicated in transplant tolerance. Mazariegos et al. have proposed using the plasmacytoid dendritic cell:myeloid dendritic cell ratio in recipient blood as a measure of tolerance, although this is controversial.^102,108,109 It would be an interesting correlate to direct innate in vitro assays to determine whether tissue-engineered constructs induce this tolerogenic dendritic cell subset in vivo.

Finally, one common method of evaluating rejection responses is to measure MHC alloantibodies in recipient blood. Clinically, this assay is performed routinely in transplant recipients. Anti-HLA antibodies have been linked particularly with chronic rejection.¹¹⁰ Methods using ELISA and flow cytometry have recently been developed that facilitate such analyses.¹¹¹ There is some evidence that tolerant patients do not have circulating anti-HLA antibodies.¹⁰⁸

Conclusions

Transplantation and biomedical devices have separately proven their clinical utility and have separately evolved refined testing methods. In the era of combination products such as tissue-engineered constructs, we must unite these techniques to allow immune-based evaluation of candidate biomaterials and ultimately to guide design that leads to immune tolerance. Examination of the effects of biomaterials on APCs will provide valuable insight into the mechanisms of response, but the complex inter-relationships between innate and adaptive immunity demand that we use more-sophisticated approaches to determine the immunological fate of tissue engineering.

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