Thermal and compositional characterization of chicken, beef, and pork cartilage to establish its lifetime

Abstract

The thermal behavior of commercial chicken, beef, and pork cartilage, were studied using thermal analysis techniques. We use thermogravimetry (TGA) to study their thermal stability between room temperature and 500 °C; differential scanning calorimetry (DSC) in a temperature range between - 50 °C and 300 °C to determine their phase changes associated with endothermic or exothermic processes, and mass spectrometry coupled to TGA to determine the release of elements as they are heated; the results are similar for the three samples.

In the thermogravimetric analysis, three different phases were found corresponding to the stages of dehydration (21 °C < T < 100 °C), decomposition (100 °C < T < 300 °C, and degradation (300 °C < T < 500 °C). The DSC study shows two endothermic anomalies corresponding to melting of the aqueous content (−25 °C < T < 25 °C) and evaporation of the aqueous content (27 °C < T < 175 °C), with required enthalpies of 137.30 J/g and 1193 J/g, respectively. Mass spectrometry evidenced the release of molecules such as nitrogen, oxygen, carbon dioxide, and calcium.

This study intends to give an approximation to the possible behavior of commercial cartilage that is stored for use in surgery, in no way is it intended to simulate the behavior within the human body, since the biological and physicochemical parameters inside the body are not studied.

From the TGA results for different heating rates, we calculated the activation energies required in each of the phases, whose values are 3250,95 J/mol in the dehydration stage, 5130,63 J/mol for decomposition, and 22,677,52 J/mol for degradation. With the activation energies and following the Toops theory (TOOP, 1971) [13], we proceeded to calculate the lifetime in the completion of the three stages or what in thermogravimetric analysis, is known as useful life per stage, finding that a sample of cartilage stored under ambient conditions, after 62 days it loses its initial properties. Which provides an important parameter for the storage of possible synthetic biomaterials with properties similar to cartilage. It is clear that here the useful life or the change of the original properties due to temperature effects is studied, which under the Arrhenius theory is transferred to the kinetic study over time.

1. Introduction

Degenerative diseases related to articular cartilage wear, such as osteoarthritis, are a public health problem that mainly affects the quality of life of the population over 45 years of age [1], causing work incapacity and functional dependence when in advanced stages [[2], [3], [4], [5]].

There are many efforts made by the scientific community to understand the composition of cartilage, its function, its wear and its possible replacement, with the purpose of contributing to a solution for degenerative diseases related to this biomaterial [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]].

Worldwide, osteoarthritis is the fourth most prevalent rheumatic pathology in healthcare facilities [6]. This condition has a much greater impact on the knee joint, and is considered a progressive disease with no cure [7]. Several pharmacological and surgical treatments help to temporarily relieve pain and improve quality of life. These treatments focus on AINES, opioids, hyaluronic acid injections, and autologous chondrocyte implantation [8,9]. However, none is a definitive solution to the problem [10].

With this paper, we want to contribute to the aspect of cartilage composition, its stability, and its useful lifetime from the reaction kinetics; using thermal analysis techniques, to provide an approach to possible materials that can serve as replacement of this biomaterial in case of loss or wear.

With this work, we seek to support all scientists who are looking for a replacement solution for this biomaterial, for which we only consider behavior outside the human body, which is useful for storage conditions of possible synthetic materials, without taking into account the biological factors that they exist inside the body.

Thanks to thermal analysis, it is possible to analyze and find both the thermal behavior, composition, and physicochemical properties of materials, including biomaterials [11]. Characterization with these techniques allows us to have a solid theoretical basis to recreate the original tissue properties from different commercial materials.

2. Materials and methods

Different samples of commercial cartilage from beef, chicken and pork were obtained freely in the market place of Santa Elena in Cali - Colombia. Its characterization was carried out at the Thermal Analysis Research Laboratory of the Autonomous University of the West of Cali, Valle del Cauca-Colombia. Prior to the measurement, the samples were washed with deionized water and a thermal bath at a temperature of 30 °C for 4 h.

A helium purge gas of 50 ml/min in the sample, and 10 ml/min in the surrounding atmosphere was used in the thermal analysis equipment; this atmosphere was chosen after preliminary tests in which it was observed that the samples have nitrogen in their composition.

2.1. Obtaining hyaline cartilage samples

Chicken, beef, and pork cartilage samples, were obtained from commercial sites and washed with distilled water (Fig. 1), when sample are in the laboratory, they are sterilized, washed with deionized water, and dried in an oven at 30 °C for 2 h prior to the measurements.

Fig. 1.

Cartilage samples used in the measurement: (a) Pork sample, (b) Pork first sample, (c) Pork final sample, (d) Chicken sample and (e) Beef sample.

Before the measurements, the samples were stored refrigerated at a temperature of 1 °C for 2 h to maintain their properties intact during the characterization process. For all measurements, a sample mass in the order of 3 mg was used.

2.2. Thermal characterization techniques

The thermal properties were continuously measured as a function of temperature, with a differential scanning calorimeter (DSC Q2000, TA Instruments) using a helium atmosphere in a range of −50 °C–300 °C at a rate of 10 °C/min to quantify the energetic changes associated with the first-order phase transitions in the material at low and high temperatures. A Thermogravimetric analyzer (TGA Q500, TA Instruments) was also used to quantify the change in weight of the spider silk as it was subjected to temperature changes ranging from ambient temperature to 550 °C with a rate of 10 °C/min and under an inert helium atmosphere. Finally, a mass spectrometer (MS Discovery, TA Instruments) was used alongside thermogravimetric analysis (TGA) to identify the gases detached in the thermogravimetric analyzer, The MS measurement was made in an atmosphere of gaseous helium. Each measurement was repeated five times to achieve the highest possible precision.

2.3. Estimation of cartilage lifetime by TGA decomposition kinetics

Obtaining the useful life of a material employing thermogravimetry (TGA) is an established method for predicting the long-term aging characteristics of a material in a short experimental time, taking advantage of the activation energy (Eq. (1)) of the material, obtained through the Arrhenius equation, by varying the parameter of the heating rate [[12], [13], [14]].

$$=A{e}^{-\frac{E_a}{RT}}$$ (1)

By deriving the equation of the activation energy (Eq. (2)) with respect to the inverse of the temperature, we obtain:

$$E_a=-\frac{R}{b}\frac{dlog\beta }{d(1/T)}$$ (2)

Where:

E_a = Activation Energy (J/mol).

R = Gas Constant (8314 J/mol K).

T = Temperature at Constant Conversion (K).

β = Heating Rate (°C/min).

B = Constant (0,457).

Toops has postulated a relationship between activation energy and the estimated lifetime of some wire insulation [13] (Eq. (3)).

R: Constante universal de los gases.

E: Energía de activación.

P(X): Función que depende se la energía de activación an altas temperaturas

β: Velocidad de calentamiento promedio

$$\log t=\frac{E}{2.303RT}+\log \left(\left(\frac{E}{\beta R}\right)*P\left(\frac{E}{RT}\right)\right)$$ (3)

3. Results and discussion

3.1. Thermogravimetric analysis (TGA) for different cartilages

Fig. 2 shows the results obtained by thermogravimetry in an inert helium atmosphere for samples of chicken, beef, and pork cartilage, using about 5.0 mg of each sample and measuring between 21 °C and 500 °C. Three different stages are observed, consisting of dehydration (21 °C < T < 100 °C), decomposition (100 °C < T < 300 °C, and degradation (300 °C < T < 500 °C).

Fig. 2.

Weight behavior as a function of temperature for beef, chicken, and pork cartilage.

The thermogravimetry results indicate that chicken cartilage shows weight losses of 55.40% in the dehydration stage, 28.56% in the decomposition stage, and 3.41% in the degradation stage. The percentages of weight loss in beef cartilage were similar, obtaining losses of 55.47% for dehydration, 26.89% for decomposition, and 4.03% for degradation. On the other hand, for the pork sample, the dehydration stage generated less loss than for beef and chicken, with 41.85% loss of weight in the sample, 34.70% loss for the decomposition stage, and 7.73% loss of weight for the degradation stage.

When superimposing the TGA results for the three samples (chicken, beef, and pork), an appreciable difference is observed in the results of pork concerning beef and chicken, mainly in the dehydration stage, because in the pork sample there is a higher percentage of fat, which generates less water storage.

3.2. Differential scanning calorimetry (DSC) for cartilage

The graph in Fig. 3 shows the behavior of heat flow with temperature for commercial chicken, beef, and pork cartilage, in which two endothermic anomalies are evidenced, the first associated with the melting process of the water they contain and the second with its evaporation, this behavior is consistent with the ability of cartilage to form crystals in its structure.

Fig. 3.

Comparison of DSC curves for Beef, Chicken, and Pork cartilage.

For pork hyaline cartilage, the heat flow curve shows a first endothermic anomaly at 0.06 °C, associated with a melting process of the surface water contained in the sample, with a required energy value or associated enthalpy of 113,7 J/g, and presents a second endothermic anomaly also associated with the evaporation of the aqueous content with a required enthalpy of 1137 J/g. In the same way, endothermic anomalies were found for beef and chicken cartilage, whose information is contained in Table 1, with an additional small endothermic anomaly in chicken cartilage associated with slight molecular rearrangement.

Table 1.

Heat flow for beef, chicken, and pork cartilage.

Samples	Transition temperature (°C)		Absorbed Heat (J/g)	Process type
	Initial	Final
Beef hyaline cartilage	−25	25	137,30	Endothermic
	27	175	1193,0	Endothermic
	200	210	8699	Endothermic
Chicken hyaline cartilage	−25	13	131,20	Endothermic
	25	156,25	1037,0	Endothermic
Pork hyaline cartilage	−25	6	131,20	Endothermic
	10	140	1137	Endothermic
				Endothermic

Correlating the results obtained for TGA and DSC of the three cartilages, it can be appreciated that the dehydration obtained in the thermogravimetry technique is related to the second endothermic peak found in the DSC results. Due to the measurement range of the two equipment used, the TGA does not show the melting process of the aqueous content, although it is not associated with mass loss, and the DSC does not show the degradation process, which if it were evidenced, it would be shown as an endothermic anomaly.

3.3. Mass spectrometry (MS) analysis

The gases released in the TGA oven, as the cartilage samples were heated, passed directly to the mass spectrometer through a capillary that was maintained at 300 °C. The spectrometer delivers the value of the ionic current required to identify the ions released, these can be observed in the graphs of Fig. 4, Fig. 5, Fig. 6, wherein the left vertical axis is the percentage of weight loss and the right vertical axis the value of the ionic current required to identify the element, it is important to clarify that the mass spectra are the transversal lines and those at the top are the ones that are released first, being the last ones corresponding to the last elements released by the samples.

Fig. 4.

TGA-MS analysis for chicken cartilage.

Fig. 5.

TGA-MS analysis for beef cartilage.

Fig. 6.

TGA-MS analysis for pork cartilage.

Fig. 4, Fig. 5, Fig. 6 show the results obtained for the chicken, beef, and pork cartilage samples, respectively. In the spectrum, molecules such as nitrogen, oxygen, carbon dioxide, and calcium (identified by their mass charge ratio), which are the main components of the synovial fluid, are identified. It is observed that the three samples give off the same ions, but their concentration varies slightly, which is evidenced by the appearance of small peaks upwards and slight deviations from the baseline associated with the ion for each sample.

Table 2 shows the different elements identified in the three cartilages, together with their chemical formula, their mass charge ratio, and the temperature range in which they were detached. Knowing these elements is essential when developing possible cartilage replacements since there are currently [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]], many efforts to contribute to the regeneration of cartilage wear.

Table 2.

Synthesis of elements identified in chicken, beef, and pork cartilage samples.

Element	Formula	Mass/charge ratio	Release temperature
Water	H2O	18	25 °C–100 °C
Nitrogen	N	14	Constant release
Calcium	Ca	40	Constant release
Carbon dioxide	CO2	44	300 °C–350 °C
Monoatomic oxygen	O	16	280 °C–350 °C
Silicon	Si	29	Constant release
Sulfur	S	32	Constant release
Phosphorus	P	31	Constant release
Hydroxyl	OH	17	25 °C–90 °C, 300 °C
Helium measuring atmosphere	He	3	300 °C–490 °C
Isotope O2	Isotope O2	15	250 °C–390 °C
Isotope N2	Isotope N2	27	290 °C–350 °C
Diatomic Nitrogen	N2	28	Constant release

3.4. Evaluation of lifetime for each reaction stage

For this study, it was necessary to study the behavior of the weight of the different cartilages as a function of temperature, at different heating rates (5 °C/min, 7 °C/min, 10 °C/min, 14 °C/min, 17 °C/min, 20 °C/min). After this, all the behaviors obtained are plotted and those that are similar are kept, keeping only 4 sweeps in this case (Fig. 7, Fig. 8, Fig. 9).

Fig. 7.

Selected sweeps and measurement phases - Chicken hyaline cartilage.

Fig. 8.

Selected sweeps and measurement phases – beef Hyaline cartilage.

Fig. 9.

Selected sweeps and measurement phases – pork Hyaline cartilage.

Then, each phase was studied separately (dehydration, decomposition, and degradation), and the temperature points where the mass loss was found were tabulated for each heating rate. In addition to this, the expression 1000/T was calculated for each of the points and the logarithm of the heating rate vs 1000/T was plotted (Fig. 10), where a linearization is made and from the values of the slope the activation energy of each phase is obtained (equation (2)) and by means of its antilogarithm and according to Toops (equation (3)) the expression P(X) is found, all the results obtained are shown in Table 3.

Fig. 10.

Log β vs 1000/T plot for beef cartilage sample.

Table 3.

Activation energies obtained from plots of the logarithm of the heating rate vs 1000/T.

Phase	Mass loss (%)	Slope (a.u)	Activation energy (J/mol)	P (E/RT) (J/mol)
Dehydration	10	−1,2088	3250,95	0,2265
	15	−1,20,934
	20	−1,11,416
	25	−1,02237
	30	−1,01559
	35	−0,99,038
	40	−0,9844
	45	−0,95,543
	50	−0,9553
Decomposition	55	−1297	5130,63	0,2265
	60	−1027
	65	−0,99,421
	70	−0,9897
Degradation	75	−2723	22,677,52	0,02418
	80	−3512
	85	−3579

With the help of equation (3), the time data found for different temperatures in the analysis range are tabulated and graphs are obtained that describe the time behavior of the samples according to the temperature, helping to know the ideal storage conditions of the samples.

Fig. 11 analyzes the dehydration stage of the cartilage and shows that at temperatures below zero, there is no dehydration activity in the sample. For the chicken and beef samples, the behavior in lifetime is practically the same, both samples at 300 K would take an average time or half-life of 90 min to dehydrate, while at 450 K, they would take approximately 30 min, this for a sample of 3 mg.

Fig. 11.

Behavior of shelf life for dehydration stage.

Fig. 12 shows the analysis of the decomposition stage, in which a considerable change in time is evident. Changes in the sample begin to be observed at an average time of 1500 min (25 h) at approximately 280 K (7 °C). At approximately 300 K (30 °C), the sample, without any external variable factor, would take 700 min (11 and a half hours) to decompose, i.e. to present the release of ions previously mentioned in the mass spectrometry. It should be clarified that this time is after the dehydration stage, so the decomposition temperature conditions should take into account the time it takes for the samples to release the aqueous content.

Fig. 12.

Behavior of the shelf life for the decomposition stage.

The study of the useful lifetime for the degradation stage is presented in Fig. 13, showing a more pronounced behavior in temperature, since, at 300 K, the sample would take 41 days to leave its last residues and at 350 K it reaches the final degradation stage of the sample. The complete degradation of a sample in total must contemplate the estimated times for dehydration and decomposition since it must go through these stages to achieve degradation.

Fig. 13.

Lifetime behavior for degradation stage.

4. Conclusions

The articular cartilages of chicken, beef, and pork, as organic material, present three thermal processes as they are heated, which correspond to stages of dehydration, decomposition, and degradation. The results show that the three cartilages turn out to be practically the same allowing to have a standardization of the properties of this tissue in the face of temperature variations in a regular way.

Pork cartilage has slightly different concentrations of compounds than chicken and beef cartilage due to the fat content in its structure.

Knowing the composition of a material by mass spectroscopy, which is a technique that identifies detached ions, in conjunction with TGA and DSC techniques, presents a clearer path to identifying constituent elements and potential commercial replacement materials for use in synthetic biomaterials.

Lifetime calculations show that cartilage must be kept at a temperature below 273 K to achieve perfect preservation of the element since it dehydrates easily. Even at 273 K (0 °C), the sample decomposes after 62 days, due to its organic components. Considering the commercialization of a possible synthetic biomaterial, it is concluded that the distribution and storage time under optimal conditions of the material should be approximately 7 days.

Author contribution statement

• 1- Conceived and designed the experiments: Gladis Miriam Aparicio Rojas and Lina Juliana Andrade
• 2- Performed the experiments: Gladis Miriam Aparicio Rojas and Lina Juliana Andrade
• 3- Analyzed and interpreted the data: Gladis Miriam Aparicio Rojas and Lina Juliana Andrade
• 4- Contributed reagents, materials, analysis tools or data: Gladis Miriam Aparicio Rojas and Lina Juliana Andrade
• 5- Wrote the paper: Gladis Miriam Aparicio Rojas and Lina Juliana Andrade

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interest's statement

The authors declare that we have no conflict of interest.