The in vivo cytokine release profile following implantation
Introduction
The rapid advance of tissue engineering should allow the development of more implants for tissue regeneration or repair. To predict their success at an early stage, prior to animal implantation, it is imperative to have a full understanding of the environment in which they are to be implanted. Introduced cells can be affected either positively or negatively by protein mediators at the implant site and so identification of the profile and concentration of these markers is invaluable. To date, there have been a number of studies that have determined the presence of cytokines in tissue, many studies have analysed cell types and individual mediators of inflammation [1], [2], [3], [4] but no complete data is yet available that allows the recreation of the inflammatory environment in vitro. There is a great need in the medical fields to investigate the real-time expression of biological markers to develop the knowledge concerning their roles in implant integration and homeostasis.
Knowledge of the inflammatory response directed towards a material following implantation in vivo is imperative to determine the long-term integration, performance and survival of the material. When considering regenerative medicine applications that incorporate cells within a carrier or matrix such as a porous construct, consideration must also be given to the impact of inflammation on the cells within the material, specifically their viability, functionality and phenotype. The immune response and the resulting release of chemical mediators clearly has the well proven potential for altered cell differentiation and function, in fact it is these mediators that are often used to control differentiation of stem cells in vitro [5], [6].
Materials have been used extensively for over 50 years to replace or augment parts and functions of the human body yet the inflammatory response to these materials has not been fully elucidated. Many studies have investigated implant failure by utilising tissues from around explanted devices and these studies provide some indication of the nature of inflammation but little is known about the response short term or under more controlled conditions.
One of the most commonly used methods of studying the normal host response to an implanted material is by using an animal model. Samples of the material for evaluation are implanted to determine the surrounding tissue responses. There are a number of different formats and methodologies that have been used but these fall into two subgroups; direct implantation where a sample of the material is in direct contact with the tissue or indirectly utilising a hollow tube implantation model into which the material rests [7]. The direct implantation allows the study of the tissue response in contact with the material through the histological analysis of cell types alongside molecular analysis of message expression. The indirect hollow tube method allows for the study of the fluid phase response to materials through the collection and analysis of exudates. The advantage of this model is that it not only allows the analysis of fluid and cells close to an implanted material but also allows the collected fluid to be used for further in vitro analysis.
These different implantation models have been utilised by a number of groups to study the response to implanted materials [8], [9], [10], [11], [12], [13], [14]. For example, a study that implanted titanium and PTFE within the hollow tube model demonstrated differences in the inflammatory response with respect to cell counts and IL-1 release with PTFE generating a greater increase in total cell numbers [15], [16]. The analysis of different cell types induced by implants following implantation [17], [18] provides important data that demonstrates an initial infiltration of granulocytes that are gradually replaced by macrophages and fibroblasts that are involved in tissue remodelling. There are a limited number of studies that look at cytokine release in response to materials, specifically data is available that measures TNF-α, IL-1β, IL-1α, IL-6 and IL-10 release in response to titanium implantation [19], [20], [21] but this study was carried out at very short time points up to 48 h and so provides no information about the longer term reaction. Medium term data over time periods of 7 days is available for IL-1 production in response to silicone [22]. Although many studies have utilised the rat model to consider the soft tissue response to materials these do not provide the data for extrapolation to in vitro studies.
The hypothesis tested was that an acellular hollow chamber in a subcutaneous model provides a means to profile the host cytokine response over time. This data could then be used to determine in vitro cell function and differentiation for cell based regenerative medicine therapies using cells, stem cells, carriers or scaffolds. Polymethylmethacrylate (PMMA) was chosen because it is a versatile and proven biomaterial having successful applications in medicine for in excess of 50 years. It is commonly used for, arthroplasties, cranioplasties, and as a cement for many orthopaedic prostheses. It is also commonly used as a standard control when evaluating new materials [23]. Polymethylmethacrylate (PMMA) was the chosen material due to its successful performance in medicine without inducing, in bulk form, adverse reactions [24], [25]. The capsules for implantation were made by sealing the ends of PMMA tubing with cellulose nitrate paper, itself a well tolerated material used as a negative control in angiogenic assays [26]. The capsule was made by sealing each end of the PMMA with cellulose nitrate, another well tolerated material that has been used as a negative control in some angiogenesis studies. Overall, the capsule was expected to be relatively well tolerated and as such, could act as a baseline model for the typical non-specific response to implanted biomaterials.
The aim of this study was to measure and plot the time course of the normal inflammatory response initiated upon implantation of a biocompatible material. A hollow tube model was utilised and implanted intramuscularly in rats allowing the collection of the exudates at various time points. The cytokine levels were then determined using ELISA. The measured response and cytokine release could therefore be considered non-specific and typical of inflammation induced upon the implantation of most non-degradable constructs.
Section snippets
Chamber manufacture and implantation
Chambers for implantation and collection of exudates were prepared from 1 cm lengths of 1 cm diameter medical grade PMMA tubing (Goodfellows, UK) sealed at each end using 0.45 μm cellulose nitrate filter paper (Whatman, UK) attached using medical grade silicon glue (Nusil Silicone Technology, USA) and were sterilised by autoclaving. Four test chambers were implanted subcutaneously into individual pockets created by blunt dissection into the backs of Wistar rats, two on either side of the spine,
Results
The appearance of the exudates varied from clear to a straw yellow colour through to dark red and the volume obtained varied from 150 μl to 750 μl with increasing time. The volume, appearance and optical density values for each sample were recorded (Table 1). Standard curves were plotted for each ELISA enabling the concentrations for each cytokine in each sample to be calculated. Four values were obtained for each cytokine for each time point and the mean and standard deviations plotted (Fig. 1,
Discussion
This study demonstrated the typical inflammatory mediator release profile following implantation of a well tolerated and proven non-degradable material. The response upon implantation of a biomaterial such as PMMA is different from the simple healing response following injury. The response is termed the foreign body reaction and involves an acute phase of inflammation followed by a chronic phase that ultimately triggers healing and tissue remodelling. Neutrophils are initially present in the
Conclusions
This experimental data provided valuable information about the baseline production of cytokines upon implantation of a material. This knowledge of the specific levels of these proteins in a complex cocktail provides the specific information required to recreate the in vivo environment in an in vitro setting and may prove invaluable for studying the downstream effects of inflammation on other biochemical pathways including cell differentiation and function.
It may be possible to utilise this
Acknowledgments
The authors gratefully acknowledge the support of the joint UK Research Councils’ Interdisciplinary Research Collaboration in Tissue Engineering (BBSRC, EPSRC and MRC).
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