Originally published May 1998
TECHINICAL PAPER SERIES
Some of the earliest scientific measurements were of heat produced or absorbed during physical changes of state and chemical reactions. Crude calorimeters for this purpose were among the first scientific instruments. For example, Bunsen (18111899) placed small animals on blocks of ice, and used differences in the amount of melted water produced as a measure of relative metabolic activity.
Calorimetry has, of course, progressed steadily. However, not many biomaterials scientists are aware of the sensitivity and power of a relatively new techniqueisothermal microcalorimetry (IMC)as a tool for detecting either the rate of material-degradation processes or the magnitude of cellular metabolic responses to materials, nor is there much awareness of the predictive power of such data. This article presents the principles of IMC, along with the results of some initial studies.
DYNAMIC CHEMICAL STABILITY OF BIOMATERIALS
For a given material-degradation process or combination of processes, the net rate of heat produced or absorbed is directly proportional to the net reaction rate. In IMC, a specimen is placed in a test well under conditions of simulated use, and the net heat-flow rate between the test well and its surroundings is measured. To minimize external effects, instruments of the type used in this study (Model 4400, Calorimetry Sciences Corp., Provo, UT; see Figure 1) have test wells for which heat-flow rates are measured relative to a reference well. As little as a few microwatts of heat flow can be measured precisely and accurately. Consequently, slow reactions in amounts as small as several grams of material can be monitored.
Figure 1. Block diagram of the CSC Model 4400 isothermal microcalorimeter.
Short-term IMC rate data can also be used to predict long-term results. The validity of such predictions has been verified for solid pharmaceuticals by comparing results for conventional multiyear degradation studies with those predicted by IMC runs of a few days.1,2 In classic IMC, rates are measured at multiple temperatures to determine an activation energy.1 To determine the rate constant, k1, and to calculate the percentage of degradation, it is necessary to know a separate rate constant k2 from chemical analysis of the amount of material degraded over time at some temperature.
The need for a separate k2 has been eliminated through the use of a powerful new method for treating IMC reaction-rate data, introduced by Willson.2 Heat-flow rate is measured only at the temperature of interest, typically for ~200 hours. Using standard computer software, an equation that fits the data is determined and integrated from time 0 to (infinity) to give the complete heat content (Hc) of the specimen for the combination of processes being measured. The equation is then reintegrated over a time of interest, t, to give Ht. Then, Ht/Hc x 100 = percentage of reaction in time t.
Biomaterials Degradation. In our first study, IMC was used to predict the storage stability of pellets of implantable bone-void filler, formulated both with and without the antibiotic tobramycin. The pellets were medical-grade, fully hydrated calcium sulfate (supplied by Wright Medical Technology, Arlington, TN), designated here as CaS and CaS-tobra. For each run, 100 pellets (12.5 g total) were sealed in moist air (27% RH) in a glass ampule.
The heat-flow rate for CaS alone was essentially constant at each temperature, but became increasingly negative (endothermic) with increasing temperature (see Figure 2). At each temperature, heat flow was more exothermic for CaS-tobra than for CaS. In addition, a decrease in the CaS-tobra heat-flow rate was readily apparent at 40° and 50°C over 25 hours. The activation energy for CaS-tobra calculated from IMC data was 19.0 kcal/mole, comparable to values for similar compounds as determined by conventional techniques.1
Figure 2. Averaged heat-flow rates (n = 3) for calcium-sulfate bone-void filler, with and without tobramycin.
Heat absorption by CaS alone can be attributed to slight water loss. The amount would be virtually undetectable by techniques other than IMC, and indicates that the CaS pellets are extremely stable in air at the temperatures studied. Compared with CaS alone, CaS-tobra pellets produced heat with an activation energy attributable to tobramycin degradation. Therefore, one can assume that the exothermic difference for CaS-tobra versus CaS alone was due to degradative changes in tobramycin. From the IMC data, the predicted degradation of CaS-tobra was <0.1% per year at 30°C.
Polyethylene Chemical Stability. In our second study, IMC was used to compare the dynamic chemical stability of ultrahighmolecular weight polyethylene (UHMWPE) in moist air (30% RH) at 25°C following sterilization by four different methods: gamma irradiation in air (Air) and in nitrogen (N2), ethylene oxide gas (EtO), and gas plasma (GP). The investigations were conducted on 5 g of 2- x 6-mm-diam pellets of a reference-grade UHMWPE sealed in moist air (30% RH) in a glass ampule.
Based on other UHMWPE studies, the exothermic activity can be assumed to be mainly oxidation of various susceptible chemical bonds (see Figure 3).3 Results for GP- and EtO-sterilized UHMWPE showed no significant rate increase compared with unsterilized controls. In contrast, the rate of reaction for Air or N2 specimens indicates an approximate seven- to tenfold increase in the number of
oxidizable bonds present soon after sterilization. The difference between Air and N2 was simply the result of a delayed onset of air exposure for N2 specimens.
Figure 3. IMC data at 25°C in moist air (30% RH) for UHMWPE sterilized by different methods (n = 3).
The decline in rate for gamma-sterilized specimens indicates a change in availability of reacting species, and invited treatment of data by the Willson method. For both Air and N2, data were best fit by equations of the form
with R2 > 0.99. The predicted extent of the measured exothermic processes taking place over 1 year (% Ht/Hc) were 1.52% for Air and 1.02% for N2. The extremely slow rate makes it unlikely that there was enough oxygen depletion in 200 hours to cause the decline. A more likely cause was the increasing time necessary for oxygen to diffuse further into the UHMWPE and reach unoxidized bonds.
METABOLIC RESPONSE OF CELLS TO BIOMATERIALS
IMC has also been used to study cell metabolism in vitro.4 In 1991, Swedish researchers first applied IMC to the study of the metabolic response of cells to biomaterials.5 They compared the metabolic response of human granulocytes in vitro to three different dialysis membranes: regenerated cellulose (RC), polyether-polycarbonate (PEC), and polyacrylonitrile (AN). The control material was fluorinated ethylene-propylene (FEP) film, which was found to elicit virtually no response. Granulocytes in culture medium were placed in IMC ampules lined with the material of interest. Subsequently, zymosan particles (derived from yeast cell walls) were introduced into the ampules as a standard agent for eliciting a phagocytic response. The metabolic response to these particles was reduced if the granulocytes had already responded to the liner.
Samples in sealed ampules are lowered into the calorimeter's measurement well prior to data collection. Photo: Calorimetry Sciences Corp.
Results were expressed as heat production for 2 hours after particle introduction, where 100% was the baseline rate without particles. The values (n = 810 runs) are shown in Figure 4. As can be seen, the granulocytes had already responded strongly to RC, and less so to the other materials, in the sequence shown. The results mirror clinical measures of impairment of granulocyte function, with RC producing the most impairment and AN the least.
Figure 4. Granulocyte metabolic response to zymosan particles.5
These data stimulated us to speculate that the principle behind the Swedish studies might be extended to metabolic responses of macrophages to orthopedic wear particles. If a technique could be developed, it could potentially serve as an in vitro screening tool for studying the response of macrophages to various particle variables (e.g., type, size) and for related studies of biologic variables. Macrophage responses can, of course, be evaluated from amounts of cytokines produced, but assay choices must be made. For screening purposes, we believe that measuring the overall metabolic response has merit. Together with the instrument manufacturer, we received an NIH grant in September 1997 to pursue this line of investigation. Thus far, we have had initial success in monitoring the basic metabolic rate of IC 21transformed mouse peritoneal macrophages. We have also designed and built special IMC chambers for the macrophage/wear-particles studies, and are successfully measuring metabolic response.
This initial work suggests that IMC is a promising, sensitive technique for evaluating and predicting the dynamic chemical stability of biomaterials, and for measuring in vitro the metabolic responses of living cells to various biomaterials in solid and particulate form.
The authors would like to acknowledge the contribution of Johnson & Johnson Professional, Inc. (Raynham, MA) in providing funds for IMC instrument purchase. Additional project support was provided by Wright Medical (Arlington, TN) and through an NIH NIAMSD grant #1R41-AR44581-01. An earlier version of this study was presented at the 17th Southern Biomedical Engineering Conference, held in San Antonio, TX, February 68, 1998.
1. Koenigbauer MJ, Brooks SH, Rullo G, et al., "Solid-State Stability Testing of Drugs by Isothermal Microcalorimetry," Pharma Res, 9:939944, 1992.
2. Willson RJ, Beezer AE, Mitchell JC, et al., "Determination of Thermodynamic and Kinetic Parameters from Isothermal Conduction Microcalorimetry: Applications to Long-Term Reaction Studies," J Phys Chem, 99:71087113, 1995.
3. Sanford WM, Moore WC, McNulty D, et al., "Hip Simulator Study of the Effect of Sterilization and Oxidation on UHMWPE Wear," Trans Ortho Res Soc, 22:95, 1997.
4. Thoren SA, Monti M, and Holma B, "Heat Conduction Microcalorimetry of Overall Metabolism in Rabbit Alveolar Macrophages in Monolayers and Suspensions," Biochim et Biophys Acta, 1033:305310, 1990.
5. Ikomi-Kumm J, Ljunggren L, Lund U, et al., "Microcalorimetric Evaluation of Blood Compatibility of Hemodialysis Membranes," Blood Purif, 9:177181, 1991.
A. U. (Dan) Daniels, PhD, is the Wilhelm Endowed Professor of Orthopaedic Surgery and director of laboratory research for orthopaedic surgery at the University of Tennessee-Campbell Clinic (Memphis, TN). He is also a professor of biomedical engineering at the University of Tennessee-Memphis, and has spent most of his career in the development and evaluation of biomaterials and orthopaedic implants. Steven J. Charlebois is a graduate student in the University of Tennessee/University of Memphis joint program in biomedical engineering. He holds degrees in materials science/metallurgy/mechanics and in biomedical engineering. Edwin A. Lewis, PhD, is executive vice president and chief technical officer at Calorimetry Sciences Corp. (Provo, UT). Before joining the company he was a full-time faculty member in chemistry at the University of Alabama and Brigham Young University.