Biochemical and Mechanical Impact of Storage Techniques on Ovine Temporomandibular Joint Discs

Biochemical and Mechanical Impact of Storage Techniques on Ovine Temporomandibular Joint Discs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biochemical and Mechanical Impact of Storage Techniques on Ovine Temporomandibular Joint Discs Daniela Trindade, Cecília R. C. Calado, João C. Silva, Ana C. Maurício, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4964539/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The temporomandibular joint disc plays a fundamental role in daily activities, and when it is compromised, severely disturbs oral health and quality of life. Decellularization is gaining interest in tissue engineering (TE) applications, but requires maintaining the native structure and composition to mimic human disc properties. This study characterizes the native ovine disc and compares conservation protocols to preserve its morphology, biochemical content (sulfated glycosaminoglycans, total and soluble collagen), and mechanical and thermal behavior. Three storage protocols were tested: (i) freezing at -20°C in phosphate-buffered saline (PBS) and thawing at 4°C (PBS + 4°C); (ii) freezing at -20°C in PBS and thawing at room temperature (RT) (PBS + RT); and (iii) wrapping the discs in PBS-embedded gauze, freezing at -20°C, and thawing at RT (Gauze + RT). Protocols were evaluated at 1, 7, and 14 days, and compared with a native disc, and a collagenase-treated discs. All conservation protocols caused changes, though less pronounced than degradation. The PBS + 4°C and PBS + RT protocols maintained original morphology, yet highlighted, a contrasting biochemical and mechanical outcome based on the thawing method. Thermal analysis revealed collagen structure changes within the first 7 days of freezing. The Gauze + RT protocol showed no significant biochemical changes over time, but the disc became dehydrated and with a higher compression modulus. For TE approaches involving decellularization, it is crucial to consider these alterations. For powdered tissue applications, the Gauze + RT method for 14 days is recommended due to minimal structural impact. Temporomandibular joint disc Ovine model Biochemical composition Freezing time storage Extracellular matrix Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The temporomandibular joint (TMJ) is a ginglymoarthrodial joint that comprises a fibrocartilaginous disc between the mandible condyle and the glenoid fossa-eminence articular complex of the temporal bone [ 1 ], where the disc is crucial for the absorption of the loads. It presents notorious morphological variations and can be divided into the anterior, intermediate, and posterior regions (in the anteroposterior dimension). The intermediate region also presents variations, which can still be divided into medial, central, and lateral (in the mediolateral dimension). These differences are reflected in the content of cells (mainly fibroblasts), collagen type I and glycosaminoglycans (GAGs), the major components of the TMJ disc [ 2 , 3 ]. The TMJ is highly predisposed to suffer from trauma or degenerative events that may lead to deviations or disorders in the condyle-disc complex, which are characterised as TMJ dysfunctions (TMDs) [ 4 ]. TMDs are present in 31% of adults and elderly and 11% of children and adolescents [ 5 ] and highly impact the psychological and social experiences of patients [ 6 ]. The dysfunctions that affect the disc include thinning, perforation and displacement from the native position [ 7 ]. Ultimately, these pathological changes can lead to more severe degenerative conditions, such as osteoarthritis and osteoarthrosis [ 8 ]. TMDs can be managed with medications, physiotherapy, and occlusal splints at the beginning of the disease. In a mid-stage, minimally invasive procedures are considered as intra-articular injections, arthrocentesis, and arthroscopy. Unfortunately, these methods lack the capacity to restore a damaged disc, and there is no consistently effective treatment or consensus on treatment choices [ 9 ]. For example, a meta-analysis showed that platelet-rich plasma (PRP) injections provide lower pain levels than hyaluronic acid (HA), and either of these injections is better after arthroscopy [ 10 ]. Interestingly, PRP is as effective as arthrocentesis [ 11 ], HA/corticosteroids and arthrocentesis [ 12 ]. Discectomy, total disc removal, is largely used in more advanced cases of TMDs. However, despite helping to restore the mandibular movements and minimise the pain [ 13 , 14 ], it does not prevent degenerative events, such as ankylosis [ 15 ]. TE is a promising area for the development of novel therapies for disc dysfunctions. However, despite the similar incidence of knee and TMJ osteoarthritis, the latter lacks research, funding and specialised doctors [ 16 ]. One promising strategy might be the use of decellularized tissues since they can recreate a highly biomimetic microenvironment, recapitulating the main morphological, structural and biochemical features of native TMJ tissue [ 3 , 17 ]. It is critical to define the animal model for the decellularisation process. Different authors have investigated different animal models. Pig and minipig are suggested as the most suitable ones since they closely resemble the human anatomy and alimentary diet [ 18 – 20 ]. However, access to the disc in these animal models is obstructed by the zygomatic arch, which covers the lateral aspect of the joint. In addition, such discs can be more difficult and expensive to obtain from a local abattoir [ 20 , 21 ], preventing their standard use in pre-clinical research. On the other hand, the ovine model, like sheep and goats, is easy to obtain, inexpensive, and has an accessible surgical site [ 21 ]. In addition, compared to humans, they are similar in shape and structure, and different authors have already demonstrated their suitability for TMJ research [ 22 – 26 ]. Another relevant point to be taken into consideration when biological materials are used is their preservation, as sometimes it is not possible to test/use them immediately after extraction. Thus, the study of freezing the discs for their conservation is still debatable; in the literature, only two studies address this subject. Allen & Athanasiou investigated from one to five freezing and thawing cycles at -20°C during 6–18 h followed by thawing at room temperature (RT) and observed no changes in the mechanical behaviour of the hogs TMJ discs [ 27 ]. However, Calvo-Gallego et al. showed that freezing the discs for more than 30 days impacted their viscoelastic properties [ 28 ]. Despite this, in both studies, only the intermediate/central zone of the disc was analysed, and the animal model chosen was the pig. This work aimed to characterise the ovine disc and evaluate the impact of several conservation protocols on the disc's native properties, including disc morphology, biochemical composition, and thermal and mechanical properties. The impact of the conservation protocols on the properties of the disc was analysed in comparison to a collagenase-treated disc. Overall, this study intends to serve as a base for future applications of this material in the TE field, especially focused on TMJ regeneration. 2. Materials and Methods 2.1 Tissue preparation TMJ discs were obtained in local butchers and dissected from lambs’ joints (7 to 15 months), in which the ligaments and retrodiscal tissues were carefully removed. All samples were morphologically evaluated in weight with a scale, in thickness with a digital calliper, and the mediolateral and anteroposterior dimensions were determined using the software ImageJ version 1.54d (NIH, Bethesda, MD, USA). Native discs were stored in 0.01 M phosphate-buffered saline (PBS) (pH = 7.4) at 4°C until further use. 2.2 Storage conditions After extraction, three storage conditions were tested: (i) freezing the discs at -20°C in PBS and thawing at 4°C (PBS + 4°C_X days), (ii) freezing the discs at -20°C in PBS and thawing at RT (PBS + RT_X days) and (iii) wrapping the discs in a PBS embedded gauze and freezing at -20°C followed by thawing at RT in PBS (Gauze + RT_X days). Moreover, different time intervals (X) were assessed: 1, 7 and 14 days. Discs were subjected to characterisation immediately after thawing. 2.3 Enzymatic digestion TMJ discs were subjected to degradation with collagenase type II enzyme (C2-BIOC, Sigma-Aldrich) in agitation at 37°C for 9h, according to Fazaeli et al. [ 29 ], to compare the results and observe its impact on the collagen fibres. Native and collagenase-treated discs were lyophilised at 0.2 mbar for 24h in a freeze-drier (LyoQuest, Telstar, Japan) before further characterisation. Afterwards, for the native disc, the five regions of the disc were separated. For the disc subjected to collagenase, once it had become a thin membrane, separating it into the five regions was impossible, so it was decided to cut out a random portion of the disc. 2.4 Water content Water content for each disc’s region and the disc subjected to collagenase was calculated with the difference in weight obtained before and after lyophilisation using the following equation: Water content (%) \(\:=\frac{\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}-\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}{\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}\times\:100\) 2.5 DNA quantification assay To evaluate the cellularity of the native discs, the DNA was extracted using the DNeasy blood and tissue kit (Quiagen, EUA), where weighted lyophilised samples were digested in proteinase K (Quiagen) at 56°C overnight. Quantification was performed with Quant-iT PicoGreen Kit (Invitrogen, Fisher Scientific, Finland), where fluorescence was measured at an excitation/emission wavelength of 480/520 nm using a microplate reader (FLUOstar Omega, BMG LabTech, Offenburg, Germany) and a standard curve of λ-DNA was used. Five samples (n = 5) were used for each experimental group and normalised to the discs' dry weight (DW). 2.6 Sulphated glycosaminoglycans quantification Sulphated GAGs quantification was performed in the native and the discs that underwent degradation, according to Silva et al. [ 30 ], using the 1,9-dimethylmethylene blue (DMMB) assay. Briefly, weighted lyophilised samples were digested in a 100 µg/ml papain (from papaya latex, Sigma-Aldrich) solution at 60°C overnight, followed by combination with the DMMB solution (Sigma-Aldrich) for 5 min at RT in the dark. A standard curve prepared using chondroitin 6-sulfate (sodium salt from shark cartilage, Sigma-Aldrich) was used, and the absorbance was measured at 525 nm using a microplate reader (SPECTROstar Nano, BMG LabTech, Offenburg, Germany). Five samples (n = 5) were used for each experimental group and normalised to the DW of the discs. 2.7 Total and soluble collagen quantification Collagen was also quantified for the native and the discs that underwent degradation. For soluble collagen quantification, weighted lyophilised samples were digested in a solution of 0.1% pepsin (from pig gastric mucosa, Roche) in 0.01 M hydrochloric acid (HCl) at RT for 48 h. Sirius red staining assay was used for the quantification as previously described [ 31 ]. Samples were mixed with 50 µM Sirius Red (Direct red 80, Sigma-Aldrich) for 30 min at RT, followed by centrifugation at 10 000 rpm for 15 min. The pellet was dissolved in 0.5M NaOH, and the absorbance was read at 540 nm using a microplate reader (SPECTROstar Nano, BMG LabTech, Offenburg, Germany). A standard curve of collagen type I (rat tail collagen, Sigma-Aldrich) was used. Total collagen was quantified with the hydroxyproline kit (Sigma-Aldrich), where weighted lyophilised samples were digested in 0.2 mL of 6 M HCL, and the concentration was determined by the reaction of oxidised hydroxyproline with 4-(dimethylamino)benzaldehyde. The conversion of hydroxyproline to collagen was performed by multiplying by 7.52 [ 32 ]. Five samples (n = 5) were used for each experimental group, except for total collagen, where three samples (n = 3) were used. All data was normalised to the DW of the discs. 2.8 Fourier-Transform Infrared spectroscopic analysis The molecular composition of the native discs, treated with collagenase and subjected to the conservation protocols, was also analysed by Fourier-Transform Infrared (FTIR) spectroscopy using an attenuated total reflectance detection mode (Alpha FT-IR, Bruker) in the mid-infrared region (400 to 4 000 cm − 1 ), with a resolution of 4 cm − 1 and 64 scans per spectrum. All spectra were corrected in relation to an analysis conducted without the biological sample. Spectra were acquired with OPUS® software (version 6.5, Bruker), and the 5 morphological regions of the disc were analysed (n = 3). Pre-processing and processing were conducted on Matlab R2012b (Mathworks Natick, MA, USA). The second derivative spectra were based on a Savitzky-Golay filter with a second-order polynomial over a 15-point window, followed by uni-vector normalisation. Specific bands of the normalised second derivative spectra were used to estimate collagen and GAGs, as previously shown to be correlated with these compounds [ 33 – 35 ]: (i) collagen by the negative band at 1338 cm − 1 , which is attributed to the CH 2 side chains vibrations, (ii) sulphated GAGs by the negative band at 1052 cm − 1 , which is assigned to C-O stretching vibrations of the carbohydrate residues and SO 3 − asymmetric stretching vibrations and (iii) sulphated and non-sulphated GAGs by the negative band at 1376 cm − 1 that is related to CH 3 symmetric bending vibrations. Since the negative peaks are analysed in the second derivative, this implies that as the peak decreases, i.e. becomes more negative, the corresponding content of the target compound increases. These correlations were verified by comparison of the second derivative spectra and the non-derivative spectra. Therefore, to simplify writing, we will refer to whether the compounds content increases or decreases wherever we refer to the bands. Multivariate spectral analysis was performed by principal component analysis (PCA) and was conducted with the second derivative spectra between 800–1800 and 2800–3600 cm − 1 . Some spectrum regions were not considered to minimise noise amplification due to derivatives. 2.9 Thermogravimetric analysis (TGA) The thermal properties of the lyophilised native discs, treated with collagenase and subjected to the conservation protocols, were studied using a Simultaneous Thermal Analyser, STA 6000 (PerkinElmer, Waltham, MA, USA) under nitrogen with a flow rate of 20 mL/min in the temperature range of 30–500°C and a heating rate of 10°C/min (n = 3). 2.10 Mechanical testing under compressive stress All samples were subjected to compression tests. A texture analyser was used (TA.XTplusC, Stable Micro Systems, UK), and the assay parameters were: 1.2 mm min − 1 extension rate, 490 N load cell and the discs were compressed until a strain of at least 55%. The compression modulus was obtained from the elastic region of the graphs (n = 3). 2.11 Statistical analysis Statistical differences between samples were evaluated on GraphPad Prism 8 software. A repeated measurement two-way ANOVA with Sidak’s post-hoc test was performed for morphological analysis. A two-way ANOVA with Tukey’s post-hoc test was employed for FTIR spectroscopy analysis. For the remaining assays, such as TGA, biochemical quantifications, and mechanical tests, a one-way ANOVA was used with Tukey’s post-hoc test. All tests were calculated with a confidence interval of 95%. 3 Results 3.1 Morphological evaluation The native discs presented an anteroposterior dimension of 13.44 ± 0.65 mm, a mediolateral dimension of 23.34 ± 1.11 mm, a thickness of 1.62 ± 0.67 mm and a weight of 0.39 ± 0.03 g (Fig. 1 ). When subjected to the degradation and conservation protocols, no statistical differences were found for the anteroposterior length (Fig. 1 – Ai, Aii). For the mediolateral length (Fig. 1 – Bi, Bii), the protocols PBS + 4°C_7 days, Gauze + RT_1 and 14 days led to a decrease of 4.5%, 4.2% and 4.4%, respectively. The thickness (Fig. 1 – Ci, Cii) was the most affected feature during the degradation process, with a decrease of 37.9%. The PBS + RT_14 days and Gauze + RT_ 7 days protocols also led to thinner discs with a decrease of 11.1% and 17.3%, respectively. Ultimately, for the weight (Fig. 1 – Di, Dii), the collagenase treatment led to a decrease of 9.8%. For the gauze + RT protocol, the increase in the freezing time led to a higher percentage of weight loss: 10.9% after 1 day, 14.4% after 7 days and 17.5% after 14 days, in relation to the native disc. 3.2 Biochemical content evaluation The water, total and soluble collagen, sulphated GAGs, and DNA contents of the native discs were determined for the five regions of the disc. The extent to which disc degradation with collagenase affects the water content and the biochemical composition was also assessed (Fig. 3 ). The differences in water content (Fig. 3 -A) between the five regions of the disc were not statistically significant, varying between 72.48 and 77.08%, with the lowest and highest values being attributed to the central and anterior regions, respectively. After degradation with collagenase, the water content increased to 88.28%, with all regions presenting statistically significant differences from the native disc. Total collagen content varied from 50.70–67.71% between regions (Fig. 3 -B), and soluble collagen varied from 11.09–13.25% (Fig. 3 -C), with the anterior and central regions having the lowest content for total and soluble collagen. The opposite was found for sulphated GAGs content (Fig. 3 -D), varying from 3.59–5.35%, with the anterior and central regions having the highest content. When subjected to collagenase, on average, total collagen increased by 3.8%, soluble collagen decreased by 27.4%, and sulphated GAGs decreased by 63.2%. No statistical differences were found for the DNA content between regions, with an average of 0.016% of the disc dry weight (Fig. 3 -E). Regarding the biochemical content as analysed by FTIR spectroscopy, it was observed that the collagenase treatment led to a deviation of the band at 1052 cm − 1 , assigned to the SO 3 − asymmetric stretching vibrations of sulphated GAGs in the native disc, towards the 1058 cm − 1 position (Fig. 4 -A, B), and was associated to a statistically significant decrease of sulphated GAGs for the anterior and medial region (Fig. 4 -C). No statistical differences were observed between the conservation protocols and the native disc. The conservation protocol, PBS + RT, estimated a content decrease in the posterior region after the 14th day compared to the 1st and 7th days of freeze. For the Gauze + RT protocol, the same behaviour was observed for the anterior region. Based on the observation of the 1376 cm -1 band (Fig. 4 -D), which is assigned to CH 3 symmetric bending vibration of sulphated and non-sulphated GAGs, it was estimated a statistically significant increase of sulphated and non-sulphated GAGs content for all regions of the disc. No major statistically significant differences were observed for all the conservation protocols in relation to the native disc. For the conservation protocol PBS + 4°C, a decrease in sulphated and non-sulphated GAGs content was estimated along the conservation period for the lateral and medial regions between the 1st and 14th days of freeze. No differences were observed along the conservation time for the PBS + RT and Gauze + RT protocols. Based on the 1338 cm − 1 band (Fig. 4 -E), which is assigned to CH 2 side chain vibrations of collagen, it was estimated an increase of the collagen content after the collagenase treatment for all discs’ regions. Compared to the native disc, the conservation protocols resulted in statistically different collagen contents. The protocols leading to a higher decrease of estimated collagen content were the PBS + 4°C_14 days in the central e medial regions and PBS + RT_ 1 day in the central, medial and posterior. Analysing the protocol PBS + 4°C, a decrease in the collagen content along time was estimated, with higher significance between the 1st and 14th days of freeze for the central, lateral and posterior regions. For the PBS + RT protocol, the opposite estimations were conducted, with a collagen content increase along the conservation period, being more significant between the 1st and 14th days of freeze for all discs’ regions and between the 7th and 14th days of freeze for lateral and posterior regions. For the Gauze + RT protocols, no statistical differences were estimated along the freezing period. The PCA of FTIR spectra shows clusters of scores according to native discs and discs subjected to the collagenase treatment and the different conservation protocols, highlighting their different molecular compositions. Scores associated to PBS + 4°C and PBS + RT protocols present some overlap in the graphs at the 1st and 7th days of freezing with the scores of the native disc (Fig. 5 - A, B). Almost no overlap exists between the native disc and scores after 14 days of freezing, when the discs are frozen in PBS, highlighting the high impact of 14 days of freezing on the sample’s molecular composition. As for the Gauze + RT protocol (Fig. 5 - C), after 7 days of freezing, there is a significant impact on the sample’s molecular composition in relation to the native disc since no overlap between the scores of these types of samples was observed. The scores from the collagenase-treated disc are the ones further apart in space in relation to all other scores, pointing to a very different biochemical composition in relation to the native disc and in relation to any of the conservation protocols. 3.3 Thermal analysis The TGA and the derivative of the TGA (DTGA) show differences between the several experimental groups tested (Fig. 6 and Table 1 ). For analysis of the TGA results (Fig. 6 -A and Table 1 ), the dehydration phase (WL 1 : 30°C < T < 100°C), the decomposition phase (WL 2 : 100°C < T < 300°C) and the degradation phase (WL 3 : 300°C < T < 500°C) were considered. From the DTGA, the denaturation peak of the hydrated collagen (TP 1 ), the peak that corresponds to the conformational changes of the collagen molecule from a triple helix structure to the random coil (TP 2 ), and the peak that corresponds to the bulk degradation of the collagen fibrils (TP 3 ) were also evaluated [ 36 , 37 ]. The native disc shows a weight loss of 11.60% in WL 1 , 21.89% in WL 2 and 41% in WL 3 . When degraded with collagenase, it releases a greater amount of water - WL 1 (15.90%) and consequently reduces its weight loss during the degradation phase - WL 3 (35.47%). For the conservation protocols, the samples were mainly affected in the decomposition phase - WL 2 . For the two protocols where the freezing is in PBS, the weight loss percentage is significantly lower on the 1st day, followed by a continuous increase until the 7th day and then until the 14th day, where the values are similar to the native disc. Analysing each protocol individually, this increase in weight loss shows that the 14th day is significantly different from days 1 and 7. The opposite is observed for Gauze + RT, in which on the 1st day of freezing the value is statistically significantly higher than the native disc, but with continued freezing, on days 7 and 14, the value is close to the ones observed for the native disc. This decrease in value also makes days 1 and 14 statistically different. Regarding the thermal peaks observed in the DTGA (Fig. 6 -B and Table 1 ), the native disc presented three occurrences: TP 1 at 63.10°C, TP 2 at 237.37°C and TP 3 at 317.20°C. When the disc is degraded with collagenase, both the TP 1 and TP 2 appear at lower temperatures, being significantly different from the native disc. For the conservation protocols, the major differences are found in the TP 2 : the two protocols where the discs were frozen in PBS, during the 1st and 7th day, the peak is non-existent, but at the 14th of freezing, the value is similar to the native disc. As for the Gauze + RT protocol, the peak appears at lower temperatures on the 1st and 7th day. Moreover, in this protocol, the degradation peak - TP 3 occurs at lower temperatures when the disc is frozen for 1 day. Analysing this protocol individually, statistical differences are also found between the different freezing times for TP 1 and TP 2 , in which increasing time leads to an increase in the temperatures. Table 1 Mean and standard deviation values of the weight loss between 30–100°C (WL 1 ), 100–300°C (WL 2 ) and 300–500°C (WL 3 ) and the thermal peaks (TP 1 , TP 2 and TP 3 ) obtained from the TGA and DTGA analysis for the native, collagenase-treated and subjected to storage discs. Statistically significant differences were calculated with one-way ANOVA with Tukey’s post-hoc test and differences represented by * p < 0.05, ** p < 0.01 and *** p < 0.001 are compared with the native disc, and represented by # p < 0.05, ## p < 0.01 and ### p < 0.001 are compared within the same conservation protocol (N = 3). WL 1 (%) WL 2 (%) WL 3 (%) TP 1 (°C) TP 2 (°C) TP 3 (°C) Native 11.6 0 ± 0.75 21.89 ± 0.97 41.55 ± 1.63 63.10 ± 0.88 237.37 ± 2.50 318.20 ± 0.83 Collagenase 15.90 ± 1.52 * 20.07 ± 0.86 35.47 ± 0.46 *** 57.37 ± 1.81 ** 224.12 ± 1.50 *** 320.07 ± 1.26 PBS + 4°C_1day 15.23 ± 1.21 16.03 ± 0.75 *** , ## 40.66 ± 1.19 63.57 ± 0.22 - 320.71 ± 2.23 PBS + 4°C_7days 15.69 ± 1.14 17.05 ± 1.19 *** , # 38.11 ± 0.63 64.75 ± 1.12 - 317.02 ± 1.48 PBS + 4°C_14days 12.19 ± 0.65 20.26 ± 0.60 ##, # 38.91 ± 0.79 61.58 ± 0.66 233.98 ± 3.21 315.86 ± 0.41 PBS + RT_1day 15.89 ± 0.34 * 16.19 ± 0.28 *** , ## 39.56 ± 1.26 63.42 ± 1.34 - 313.31 ± 1.31 ## PBS + RT_7days 14.45 ± 1.03 17.24 ± 0.78 ** , # 40.71 ± 0.57 63.57 ± 1.84 - 317.83 ± 1.00 PBS + RT_14days 12.50 ± 0.87 20.88 ± 1.23 ##, # 39.33 ± 1.29 60.61 ± 0.99 230.98 ± 1.63 321.92 ± 3.63 ## Gauze + RT_1day 8.83 ± 0.57 25.45 ± 2.20 * , # 37.86 ± 2.47 * 59.02 ± 2.24 # 216.25 ± 4.46 *** , ##. ### 310.70 ± 2.98 * Gauze + RT_7days 10.60 ± 3.30 22.85 ± 1.34 38.46 ± 1.31 60.95 ± 2.47 226.88 ± 0.68 ** , ##, # 316.29 ± 1.92 Gauze + RT_14days 11.80 ± 1.58 21.79 ± 0.58 # 39.62 ± 0.91 63.85 ± 2.20 # 234.36 ± 2.62. ###, # 314.52 3.99 3.4 Mechanical Behaviour under Compressive Loading Regarding the mechanical behaviour evaluation, the native disc presents a compressive modulus of 2.36 ± 0.07 MPa, and after the collagenase treatment, the compression modulus decreased 70% to 0.72 ± 0.04 MPa. For the PBS + 4°C protocol, during the 1st and 7th days of freezing, there was a 28% and 32% increase in the modulus, respectively, followed by a stabilisation towards the native disc values at 14 days (Fig. 7 -A, D). The opposite was observed for the PBS + RT protocol, in which the modulus remained the same for the 1st and 7th days but increased by 23% after 14 days. For these two protocols, the difference between freezing for 1 or 7 to 14 days is also statistically significant (Fig. 7 -B, D). Finally, for the Gauze + RT protocol, the modulus increased by 27% after 14 days of frozen (Fig. 7 -C, D). 4 Discussion The characterisation of animal-derived tissues is extremely important to assess their potential to regenerate or replace human tissues. Different studies have evaluated different animal models and reported that the ovine model is suitable for TMJ research [ 22 – 26 ]. However, detailed information on its DNA, water and biochemical contents, particularly in terms of its soluble collagen, and a subsequent comparison with a degraded disc has not yet been evaluated. Furthermore, an optimal conservation protocol is yet to be established. In the current work, it was observed average values for native ovine TMJ disc were 75.5 ± 2.9% water, 59.2 ± 2.4% total collagen, 11.9 ± 0.7% soluble collagen, 4.3 ± 0.3% sulphated GAGs, and 0.016 ± 0.002% DNA by dry weight. These values are similar to the human TMJ discs [ 38 ]. Interestingly, it was also found that the anterior and central regions presented the lowest collagen content and the highest sulphated GAGs content for the ovine TMJ disc. The remaining regions display the inverse correlation. Interestingly, this inverse correlation between the amount of sulphated GAGs and type II collagen is usually found in the different zones of articular cartilage [ 39 ]. Collagenase treatment has a detrimental effect on the collagen network by cleaving the chains of the triple helix into small fragments and enabling the subsequent action of other enzymes, such as gelatinases [ 40 – 43 ]. These fragmentations have been observed in internal derangements of the human disc [ 44 ], so the use of this enzyme was important to assess the extent to which the conservation protocols damage the TMJ disc. The degraded native disc turned into a gel-like structure, which consequently led to a decrease in thickness and weight. In addition, as expected, the water content increased significantly since in osteoarthritic samples this also occurs due to the breakdown of the collagen network [ 45 , 46 ]. The total and soluble collagen contents increased and decreased, respectively. Despite the increase in total collagen, this was only found for the anterior and central regions. This may be due to the portion of the disc analysed since, after freeze-drying, the disc remained in a paper-like structure, making it impossible to distinguish and divide the five regions. So, in practice, collagenase treatment can lead to a stabilisation or increase in total collagen. This phenomenon is attributed to the limited efficacy of collagenases in cleaving insoluble collagen, which is characterised by a higher degree of crosslinked fibres [ 47 ]. Interestingly, it has also been reported that subjecting insoluble collagen type II to gastric pepsin results in an elevated degree of crosslinked aggregation within the collagen fibres, while for soluble collagen, the fibres almost disappear [ 48 ]. It should be noted that collagenase treatment, by leading to the increase of total collagen, may negatively affect cellular behaviour during tissue remodelling by leading to an increase of total collagen [ 49 ]. Collagenase treatment also led to a significant decrease of sulphated GAGs, most probably due to the disintegration of the collagen network, resulting in GAGs release [ 46 ]. This is concordant with Fazaeli et al. [ 29 ] observation of a decrease in sulphated GAGs despite not finding a statistically significant reduction in collagen. The authors attributed this low reduction to the time and concentration of the enzyme. Compared to the present study, the animal model may also have contributed to this difference, as the ovine model presents a smaller disc when compared to the porcine model [ 18 ], resulting in a much more effective action of the enzyme. Ultimately, all these changes in biochemical content led to a drastic reduction in the compressive modulus, which is in accordance with the reported for the porcine TMJ disc [ 29 , 50 ]. These changes in the collagen structure were further validated by TGA and DTGA analysis, where (i) a greater amount of water was released (WL 1 ), (ii) the weight loss in the degradation zone (WL 3 ) was significantly lower, and (iii) the peak corresponding to changes in the collagen triple helix (TP 2 ) also appeared significantly earlier. To evaluate the ECM of a cartilaginous tissue, usually diverse laborious and complex methods are conducted based on histology and biochemical testing [ 35 ]. FTIR spectroscopic analysis may present complementary information concerning the sample molecular characterisation while enabling a simple, rapid (a spectrum is typically acquired in 1 min) and economic (no expensive reagents are required) analysis [ 51 , 52 ]. The spectrum represents vibration modes of diverse functional groups and, consequently, may be used to evaluate the sample's whole molecular composition and estimate the quantity of diverse biomolecules [ 53 , 54 ]. Despite the advantages of the FTIR spectroscopy technique, it can present low specificity due to overlapping bands and even due to common bonds present in different molecules [ 55 ]. Spectra derivatives may enable the deconvolution of some overlapping bands, increasing the spectra resolution [ 56 ]. Indeed, diverse authors have used the FTIR technique to estimate collagen and GAGs in articular cartilage of steers [ 34 ], humans and bovines [ 33 ]. Based on the analysis of the second derivative spectra, it was estimated that the collagenase treatment resulted in a decrease in the content of sulphated GAGs, with an average decrease of 62.9% for all regions when compared to the native disc, which is in accordance with the results obtained with the staining methods (63.2%). This decrease was also validated by the shift of the band to a higher wavelength (1052 to 1058 cm − 1 ), as previously shown to occur when elastin content decreases [ 57 ]. For collagen, a 47.6% increase was inferred based on spectra analysis. A small part of this increase is in accordance with the 3.8% increase of total collagen as determined by the staining methods. However, most probably, based on the FTIR spectra, most of the increase results from the higher exposition of the aminoacids due to collagen fragmentation by collagenase. Thus, the increased infra-red absorption of collagen is mainly due to a significant structural and conformation change of collagen and not due to an increased quantity of collagen. This is concordant with the observations based on polarised light microscopy of the loss of structural integrity after collagenase treatment [ 29 ]. There was also an increase in content for all GAGs (sulphated and non-sulphated). Although the band at 1376 cm − 1 is generally used to quantify all GAGs, it also represents the absorption of glycoproteins [ 34 ]. Therefore, this absorption increase can also result from the significant increase absorption of glycoproteins, due to proteins' significant structural changes. Spectral PCA also reinforces this by pointing a significant alteration of the molecular composition between the native and the collagenase-treated discs. Regarding the conservation protocols, the one that most affected the morphology was the Gauze + RT, which led to a more dehydrated disc and, consequently, to its reduction in thickness and weight. Based on the ECM analysis by the second derivative, the discs composition was not significantly affected by the conservation protocols in comparison to native discs. However, some impact on the disc’s composition was observed in the spectra PCA, although it was much smaller than the observed with the collagenase treatment. With TGA and DTGA, it was possible to observe the impact on the collagen structure. Both protocols in which the discs were frozen in PBS present similar results, where for days 1 and 7 the weight loss in the decomposition phase (WL 2 ) was lower, and the decomposition peak (TP 2 ) was non-existent. Gelatine, which is composed of denatured collagen, presents the collagen fibres in random coil, meaning that the TP 2 peak was also not found [ 58 ]. Therefore, freeze and thawing caused this modification in collagen fibres. Contradictorily, for the Gauze + RT protocol, WL 2 presented a significantly higher weight loss on day 1, and for TP 2 the peak for days 1 and 7 appears at a lower temperature due to lower thermal stability, as reported to occur in tendons [ 59 ] and rat tail collagen-based hydrogels [ 60 ] upon freeze and thawing. Interestingly, day 14 showed a stabilisation of the collagen network for all protocols by presenting results similar to the native disc. It is known that protein breakdown is caused by the formation of ice crystals [ 60 ], which ultimately appear to have a greater impact on the 1st and 7th day of freezing. It was also observed by FTIR spectroscopy, on all conservation protocols, a significant impact along the freezing period. To better analyse this, Fig. 8 -A shows the average values for all protocols, simultaneously considering all disc’s regions. It was possible to observe a pattern for sulphated and non-sulphated GAGs (1376 cm − 1 ) and for collagen (1338 cm − 1 ): for the PBS + 4°C protocol, increasing freezing times leads to a decrease of these compounds, whereas for the PBS + RT protocol, an increase of these compounds was found. Regarding sulphated GAGs (1052 cm − 1 ) a decrease was observed for all conservation protocols as the freezing time increased. Mechanically, the conservation protocols also had a detrimental effect on the compressive performance of the discs. PBS + 4°C led to a stiffer disc on the 1st and 7th of freezing, while PBS + RT was on the 14th day. For Gauze + RT, Allen & Athanasiou [ 27 ] also investigated this protocol and reported that the discs can be frozen up to 5 times without altering their viscoelastic properties. However, with the same protocol but with NaCl instead of PBS, Calvo-Gallego et al. showed that the viscoelastic properties change after 30 days of freezing [ 28 ]. Although in both studies only the intermediate/central zone was analysed, and the animal model chosen was the pig, these results are in agreement with those found in the present study, as with this protocol, there were alterations in the compression capability after 14 days frozen. This alteration was defined to be due to the loss of interstitial fluid in the discs, since a more dehydrated disc was found. The mechanical performance of the TMJ disc is defined by the cooperation of the different ECM components, and it is characterised as a viscoelastic structure, as it helps to absorb stress and distribute loads on the disc, cartilage and bone components [ 50 , 61 ]. In order to find out if there is a relationship between the quantitative results from the FTIR bands and the mechanical behaviour, three graphs were plotted, in which only the average is presented for better visualisation. In Fig. 8 -B, presents the relationship between sulphated GAGs (band 1052 cm-1) and the compression modulus. It was observed that both parameters of collagenase-treated and Gauze + RT_1 days decreased, while for the PBS + 4°C_14 days and PBS + RT_1 days, they increased. However, for the remaining protocols, the decrease of sulphated GAGs content led to an increase in the stiffness of the disc. Regarding the sulphated and non-sulphated GAGs content (Fig. 8 -C), it was observed that with the increase of the biochemical content, there is also an increase in the compression modulus. The same is found in the relationship with the collagen content (Fig. 8 -D). The protocols that such pattern is not extrapolated is the PBS + 4°C_7 days for sulphated GAGs and PBS + RT_7 days for sulphated GAGs and collagen. Regarding the discs treated with collagenase, this pattern is also not found because, as explained above, the increase in biochemical content for collagenase-treated samples in the FTIR analysis is actually indicative of its fragmentation. Furthermore, through all the characterisations reported in this study, no profile similar to collagenase-treated samples was found in the discs subjected to the protocols. Although it has been reported that the collagen content is related to the tensile capacity of the TMJ discs, Detamore et al. highlighted the challenge of establishing a correlation between the content of sulphated GAGs and the compressive performance [ 62 ]. Furthermore, Willard et al. pointed that even after removing 96% of the sulphated GAGs using chondroitinase, there was no significant change in the instantaneous compressive modulus [ 63 ]. On the other hand, collagen appears to exert a substantial influence on the compressive capacity of the TMJ discs [ 29 , 64 , 65 ]. These results agree with those found in the present study, in which was found a stronger relationship between compression tests and collagen content when compared to sulphated GAG. For total GAGs, there also seems to be a high association. However, as referred above, this band seems to have influence from glycoproteins, so extrapolating that, the mechanical behaviour is related to total GAGs is more complex. In summary, for the discs frozen in PBS, it was possible to conclude that the type of thawing can result in opposite behaviours as the results of the compression tests align with those obtained for the quantification of the biochemical content. However, regarding the morphological and thermal properties, the impact of the thawing method does not appear significant as similar results were obtained. Conversely, when the thawing process is the same, different results are found across all evaluated parameters, demonstrating once again the influence of the freezing method on the disc's native characteristics. Our results suggest that all the protocols induced alterations in the native properties of the ovine disc, so in the case of using the full disc for TE strategies, these should be taken into account. However, if the disc is to be used in powder form, for example, to develop minimally invasive strategies, then we recommend storing the discs in Gauze + RT for 14 days, as the dehydration and mechanical property changes observed by the 14th day become negligible. Declarations Competing Interests: The authors declare no conflict of interest. Funding: This research was funded by the Fundação para a Ciência e a Tecnologia (FCT) for its financial support through the following projects from CDRSP: UIDB/04044/2020, UIDP/04044/2020, Associate Laboratory ARISE (LA/P/0112/2020), InnovaBIOMAS (2022.10564.PTDC); from IBB: UIDB/04565/2020, UIDP/04565/2020, and Associate Laboratory i4HB (LA/P/0140/2020); and PhD studentship: 2022.12030.BD. This research was also funded through the institutional scientific employment program-contract (CEECINST/00077/2021). Authors' contributions: Conceptualization: DT, AM, NA, CM; Methodology: DT, AM, NA, CM; formal analysis: DT, CC, JS; Writing and revising of the article: DT, CC, JS, AM, NA, CM. All authors reviewed and approved the final manuscript. Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request References Donahue RP, Hu JC, Athanasiou KA (2019) Remaining Hurdles for Tissue-Engineering the Temporomandibular Joint Disc. Trends Mol Med 25:241–256. https://doi.org/10.1016/j.molmed.2018.12.007 Acri TM, Shin K, Seol D et al (2019) Tissue Engineering for the Temporomandibular Joint. Adv Healthc Mater 8:1801236. https://doi.org/10.1002/adhm.201801236 Trindade D, Cordeiro R, José HC et al (2021) Biological Treatments for Temporomandibular Joint Disc Disorders: Strategies in Tissue Engineering. 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C. Calado","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cecília","middleName":"R. C.","lastName":"Calado","suffix":""},{"id":344214751,"identity":"d58661ac-77a5-4176-8dc7-623885517aaf","order_by":2,"name":"João C. Silva","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"C.","lastName":"Silva","suffix":""},{"id":344214752,"identity":"cbae5ee8-7b3d-4e21-8e54-5fb07228e038","order_by":3,"name":"Ana C. Maurício","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"C.","lastName":"Maurício","suffix":""},{"id":344214753,"identity":"0810bf33-4cbf-4836-b30d-51a21869945f","order_by":4,"name":"Nuno Alves","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nuno","middleName":"","lastName":"Alves","suffix":""},{"id":344214754,"identity":"617f91b8-47a9-46e3-b34a-f1e4a063e02f","order_by":5,"name":"Carla Moura","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"","lastName":"Moura","suffix":""}],"badges":[],"createdAt":"2024-08-23 13:23:36","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4964539/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4964539/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63265339,"identity":"4cd74eb4-5081-43cb-89cb-7db747e61f13","added_by":"auto","created_at":"2024-08-26 10:01:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":342112,"visible":true,"origin":"","legend":"\u003cp\u003eOvine temporomandibular joint disc and its different regions: anterior, central, medial, lateral and posterior\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/1cd3d9add9707eaff04b1824.png"},{"id":63265334,"identity":"ff82f209-457c-44b1-80f6-e5b55fccfd76","added_by":"auto","created_at":"2024-08-26 10:01:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":598696,"visible":true,"origin":"","legend":"\u003cp\u003eRelative anteroposterior (Ai) and mediolateral (Bi) dimension, thickness (Ci) and weight (Di) of the TMJ discs subjected to the collagenase and different conservation protocols: PBS + 4°C, PBS + RT, Gauze + RT, before and after the freezing storage and its correspondent percentage of increase or decrease (Aii, Bii, Cii and Dii). n=6. Statistical analysis was conducted with a two-way ANOVA with Sidak’s post-hoc test, and differences are represented by * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/d29825318b52046f43e5772a.png"},{"id":63265929,"identity":"704ac381-5da8-4c70-9325-273854079b5b","added_by":"auto","created_at":"2024-08-26 10:09:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145402,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of water (A), total collagen (B), soluble collagen (C), sulphated glycosaminoglycans (D) and DNA (E) content of the native and collagenase-treated discs. Five independent samples were used for all quantifications (n=5), except for total collagen, in which three independent samples were considered (n=3). Statistical analysis was conducted using one-way ANOVA with Tukey’s post-hoc test. Significances represented by * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 are compared with the collagenase-treated disc, and represented by # p\u0026lt;0.05, ## p\u0026lt;0.01 and ### p\u0026lt;0.001 are compared within the 5 regions of the disc.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/22c210cdf7ae58a9d3e79ed5.png"},{"id":63265336,"identity":"392baf26-4a42-460f-966a-80747146be59","added_by":"auto","created_at":"2024-08-26 10:01:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":530303,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra (A), second derivative spectra (B) and box-plot graphs of the 1052 cm\u003csup\u003e-1 \u003c/sup\u003e(C), 1376 cm\u003csup\u003e-1 \u003c/sup\u003e(D) and 1338 cm\u003csup\u003e-1 \u003c/sup\u003e(D) second derivative bands of the native, collagenase and storage protocols: PBS + 4 °C, PBS + RT and Gauze + RT. The arrow indicates the shift of the band. Statistical analysis was conducted with two-way ANOVA with Tukey’s post-hoc test, and significances represented by * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 are compared with the native disc, and represented by # p\u0026lt;0.05, ## p\u0026lt;0.01 and ### p\u0026lt;0.001 are compared within the same conservation protocol (N=3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/e45125e052ed2487fd2a0a78.png"},{"id":63265338,"identity":"e419fa6c-4365-4c5e-b538-20a22cdeae95","added_by":"auto","created_at":"2024-08-26 10:01:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217836,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal components analysis of the second derivative spectra of the native, collagenase-treated discs (A, B, C) and conservation protocols: PBS + 4 °C (A), PBS + RT (B) and Gauze + RT (C).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/031c622e676a721e391aa606.png"},{"id":63265930,"identity":"c074b391-65bc-42a9-a179-c1c861d64884","added_by":"auto","created_at":"2024-08-26 10:09:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":240127,"visible":true,"origin":"","legend":"\u003cp\u003eTGA (A) and DTGA (B) curves of the discs in the native form, collagenase-treated and subjected to the different storage protocols.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/5dda2018d33abfee42a959ec.png"},{"id":63265333,"identity":"3a189c8d-5b98-47e4-9f3e-9593aab5a7e1","added_by":"auto","created_at":"2024-08-26 10:01:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":223044,"visible":true,"origin":"","legend":"\u003cp\u003eStress-strain curves of the natives and collagenase-treated discs (A, B, C) and conservation protocols: PBS + 4 °C (A), PBS + RT (B) and Gauze + RT (C). Correspondent compressive modulus is represented in (D). Statistically significant differences were calculated with one-way ANOVA with Tukey’s post-hoc test, and differences represented by * p\u0026lt;0.05, ** p\u0026lt;0.01 and *** p\u0026lt;0.001 are compared with the native disc, and represented by ### p\u0026lt;0.001 are compared within the same conservation protocol (N=3).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/077f1fe45baf532fe5a214c3.png"},{"id":63265931,"identity":"77307bda-9000-4a20-a60e-0415bc155fd0","added_by":"auto","created_at":"2024-08-26 10:09:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":102055,"visible":true,"origin":"","legend":"\u003cp\u003eBands of the second derivative spectra associated to sulphated GAGs (1052 cm\u003csup\u003e-1\u003c/sup\u003e), sulphated and non-sulphated GAGs (1376 cm\u003csup\u003e-1\u003c/sup\u003e) and for collagen (1338 cm\u003csup\u003e-1\u003c/sup\u003e) for all the protocols implemented: PBS + 4°C, PBS + RT and Gauze + RT, regardless of the disc’s regions (A). Relationship between compression modulus and sulphated GAGs (B), sulphated and non-sulphated GAGs (C) and collagen (D) for the native disc and the discs submitted to the different conservation protocols evaluated. Only the average of the results is presented.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/2daa823f1aba6da7849315f3.png"},{"id":63266471,"identity":"077c10a2-9bb2-4581-9386-5a06f2a82c4e","added_by":"auto","created_at":"2024-08-26 10:17:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2936281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4964539/v1/9f465f06-f9a5-4761-8b8a-d40b9de626cb.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBiochemical and Mechanical Impact of Storage Techniques on Ovine Temporomandibular Joint Discs\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe temporomandibular joint (TMJ) is a ginglymoarthrodial joint that comprises a fibrocartilaginous disc between the mandible condyle and the glenoid fossa-eminence articular complex of the temporal bone [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], where the disc is crucial for the absorption of the loads. It presents notorious morphological variations and can be divided into the anterior, intermediate, and posterior regions (in the anteroposterior dimension). The intermediate region also presents variations, which can still be divided into medial, central, and lateral (in the mediolateral dimension). These differences are reflected in the content of cells (mainly fibroblasts), collagen type I and glycosaminoglycans (GAGs), the major components of the TMJ disc [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe TMJ is highly predisposed to suffer from trauma or degenerative events that may lead to deviations or disorders in the condyle-disc complex, which are characterised as TMJ dysfunctions (TMDs) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TMDs are present in 31% of adults and elderly and 11% of children and adolescents [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and highly impact the psychological and social experiences of patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The dysfunctions that affect the disc include thinning, perforation and displacement from the native position [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ultimately, these pathological changes can lead to more severe degenerative conditions, such as osteoarthritis and osteoarthrosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTMDs can be managed with medications, physiotherapy, and occlusal splints at the beginning of the disease. In a mid-stage, minimally invasive procedures are considered as intra-articular injections, arthrocentesis, and arthroscopy. Unfortunately, these methods lack the capacity to restore a damaged disc, and there is no consistently effective treatment or consensus on treatment choices [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For example, a meta-analysis showed that platelet-rich plasma (PRP) injections provide lower pain levels than hyaluronic acid (HA), and either of these injections is better after arthroscopy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Interestingly, PRP is as effective as arthrocentesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], HA/corticosteroids and arthrocentesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Discectomy, total disc removal, is largely used in more advanced cases of TMDs. However, despite helping to restore the mandibular movements and minimise the pain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it does not prevent degenerative events, such as ankylosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTE is a promising area for the development of novel therapies for disc dysfunctions. However, despite the similar incidence of knee and TMJ osteoarthritis, the latter lacks research, funding and specialised doctors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. One promising strategy might be the use of decellularized tissues since they can recreate a highly biomimetic microenvironment, recapitulating the main morphological, structural and biochemical features of native TMJ tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is critical to define the animal model for the decellularisation process. Different authors have investigated different animal models. Pig and minipig are suggested as the most suitable ones since they closely resemble the human anatomy and alimentary diet [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, access to the disc in these animal models is obstructed by the zygomatic arch, which covers the lateral aspect of the joint. In addition, such discs can be more difficult and expensive to obtain from a local abattoir [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], preventing their standard use in pre-clinical research. On the other hand, the ovine model, like sheep and goats, is easy to obtain, inexpensive, and has an accessible surgical site [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, compared to humans, they are similar in shape and structure, and different authors have already demonstrated their suitability for TMJ research [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother relevant point to be taken into consideration when biological materials are used is their preservation, as sometimes it is not possible to test/use them immediately after extraction. Thus, the study of freezing the discs for their conservation is still debatable; in the literature, only two studies address this subject. Allen \u0026amp; Athanasiou investigated from one to five freezing and thawing cycles at -20\u0026deg;C during 6\u0026ndash;18 h followed by thawing at room temperature (RT) and observed no changes in the mechanical behaviour of the hogs TMJ discs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, Calvo-Gallego et al. showed that freezing the discs for more than 30 days impacted their viscoelastic properties [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Despite this, in both studies, only the intermediate/central zone of the disc was analysed, and the animal model chosen was the pig.\u003c/p\u003e \u003cp\u003eThis work aimed to characterise the ovine disc and evaluate the impact of several conservation protocols on the disc's native properties, including disc morphology, biochemical composition, and thermal and mechanical properties. The impact of the conservation protocols on the properties of the disc was analysed in comparison to a collagenase-treated disc. Overall, this study intends to serve as a base for future applications of this material in the TE field, especially focused on TMJ regeneration.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Tissue preparation\u003c/h2\u003e \u003cp\u003eTMJ discs were obtained in local butchers and dissected from lambs\u0026rsquo; joints (7 to 15 months), in which the ligaments and retrodiscal tissues were carefully removed. All samples were morphologically evaluated in weight with a scale, in thickness with a digital calliper, and the mediolateral and anteroposterior dimensions were determined using the software ImageJ version 1.54d (NIH, Bethesda, MD, USA). Native discs were stored in 0.01 M phosphate-buffered saline (PBS) (pH\u0026thinsp;=\u0026thinsp;7.4) at 4\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Storage conditions\u003c/h2\u003e \u003cp\u003eAfter extraction, three storage conditions were tested: \u003cem\u003e(i)\u003c/em\u003e freezing the discs at -20\u0026deg;C in PBS and thawing at 4\u0026deg;C (PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_X days), \u003cem\u003e(ii)\u003c/em\u003e freezing the discs at -20\u0026deg;C in PBS and thawing at RT (PBS\u0026thinsp;+\u0026thinsp;RT_X days) and \u003cem\u003e(iii)\u003c/em\u003e wrapping the discs in a PBS embedded gauze and freezing at -20\u0026deg;C followed by thawing at RT in PBS (Gauze\u0026thinsp;+\u0026thinsp;RT_X days). Moreover, different time intervals (X) were assessed: 1, 7 and 14 days. Discs were subjected to characterisation immediately after thawing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Enzymatic digestion\u003c/h2\u003e \u003cp\u003eTMJ discs were subjected to degradation with collagenase type II enzyme (C2-BIOC, Sigma-Aldrich) in agitation at 37\u0026deg;C for 9h, according to Fazaeli et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], to compare the results and observe its impact on the collagen fibres.\u003c/p\u003e \u003cp\u003eNative and collagenase-treated discs were lyophilised at 0.2 mbar for 24h in a freeze-drier (LyoQuest, Telstar, Japan) before further characterisation. Afterwards, for the native disc, the five regions of the disc were separated. For the disc subjected to collagenase, once it had become a thin membrane, separating it into the five regions was impossible, so it was decided to cut out a random portion of the disc.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Water content\u003c/h2\u003e \u003cp\u003eWater content for each disc\u0026rsquo;s region and the disc subjected to collagenase was calculated with the difference in weight obtained before and after lyophilisation using the following equation:\u003c/p\u003e \u003cp\u003eWater content (%) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{\\text{w}\\text{e}\\text{t}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}-\\text{d}\\text{r}\\text{y}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}}{\\text{w}\\text{e}\\text{t}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}}\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 DNA quantification assay\u003c/h2\u003e \u003cp\u003eTo evaluate the cellularity of the native discs, the DNA was extracted using the DNeasy blood and tissue kit (Quiagen, EUA), where weighted lyophilised samples were digested in proteinase K (Quiagen) at 56\u0026deg;C overnight. Quantification was performed with Quant-iT PicoGreen Kit (Invitrogen, Fisher Scientific, Finland), where fluorescence was measured at an excitation/emission wavelength of 480/520 nm using a microplate reader (FLUOstar Omega, BMG LabTech, Offenburg, Germany) and a standard curve of λ-DNA was used. Five samples (n\u0026thinsp;=\u0026thinsp;5) were used for each experimental group and normalised to the discs' dry weight (DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sulphated glycosaminoglycans quantification\u003c/h2\u003e \u003cp\u003eSulphated GAGs quantification was performed in the native and the discs that underwent degradation, according to Silva et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], using the 1,9-dimethylmethylene blue (DMMB) assay. Briefly, weighted lyophilised samples were digested in a 100 \u0026micro;g/ml papain (from papaya latex, Sigma-Aldrich) solution at 60\u0026deg;C overnight, followed by combination with the DMMB solution (Sigma-Aldrich) for 5 min at RT in the dark. A standard curve prepared using chondroitin 6-sulfate (sodium salt from shark cartilage, Sigma-Aldrich) was used, and the absorbance was measured at 525 nm using a microplate reader (SPECTROstar Nano, BMG LabTech, Offenburg, Germany). Five samples (n\u0026thinsp;=\u0026thinsp;5) were used for each experimental group and normalised to the DW of the discs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Total and soluble collagen quantification\u003c/h2\u003e \u003cp\u003eCollagen was also quantified for the native and the discs that underwent degradation. For soluble collagen quantification, weighted lyophilised samples were digested in a solution of 0.1% pepsin (from pig gastric mucosa, Roche) in 0.01 M hydrochloric acid (HCl) at RT for 48 h. Sirius red staining assay was used for the quantification as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Samples were mixed with 50 \u0026micro;M Sirius Red (Direct red 80, Sigma-Aldrich) for 30 min at RT, followed by centrifugation at 10 000 rpm for 15 min. The pellet was dissolved in 0.5M NaOH, and the absorbance was read at 540 nm using a microplate reader (SPECTROstar Nano, BMG LabTech, Offenburg, Germany). A standard curve of collagen type I (rat tail collagen, Sigma-Aldrich) was used.\u003c/p\u003e \u003cp\u003eTotal collagen was quantified with the hydroxyproline kit (Sigma-Aldrich), where weighted lyophilised samples were digested in 0.2 mL of 6 M HCL, and the concentration was determined by the reaction of oxidised hydroxyproline with 4-(dimethylamino)benzaldehyde. The conversion of hydroxyproline to collagen was performed by multiplying by 7.52 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFive samples (n\u0026thinsp;=\u0026thinsp;5) were used for each experimental group, except for total collagen, where three samples (n\u0026thinsp;=\u0026thinsp;3) were used. All data was normalised to the DW of the discs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Fourier-Transform Infrared spectroscopic analysis\u003c/h2\u003e \u003cp\u003eThe molecular composition of the native discs, treated with collagenase and subjected to the conservation protocols, was also analysed by Fourier-Transform Infrared (FTIR) spectroscopy using an attenuated total reflectance detection mode (Alpha FT-IR, Bruker) in the mid-infrared region (400 to 4 000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 64 scans per spectrum. All spectra were corrected in relation to an analysis conducted without the biological sample. Spectra were acquired with OPUS\u0026reg; software (version 6.5, Bruker), and the 5 morphological regions of the disc were analysed (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003ePre-processing and processing were conducted on Matlab R2012b (Mathworks Natick, MA, USA). The second derivative spectra were based on a Savitzky-Golay filter with a second-order polynomial over a 15-point window, followed by uni-vector normalisation. Specific bands of the normalised second derivative spectra were used to estimate collagen and GAGs, as previously shown to be correlated with these compounds [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]: \u003cem\u003e(i)\u003c/em\u003e collagen by the negative band at 1338 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is attributed to the CH\u003csub\u003e2\u003c/sub\u003e side chains vibrations, \u003cem\u003e(ii)\u003c/em\u003e sulphated GAGs by the negative band at 1052 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is assigned to C-O stretching vibrations of the carbohydrate residues and SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e asymmetric stretching vibrations and \u003cem\u003e(iii)\u003c/em\u003e sulphated and non-sulphated GAGs by the negative band at 1376 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that is related to CH\u003csub\u003e3\u003c/sub\u003e symmetric bending vibrations. Since the negative peaks are analysed in the second derivative, this implies that as the peak decreases, i.e. becomes more negative, the corresponding content of the target compound increases. These correlations were verified by comparison of the second derivative spectra and the non-derivative spectra. Therefore, to simplify writing, we will refer to whether the compounds content increases or decreases wherever we refer to the bands.\u003c/p\u003e \u003cp\u003eMultivariate spectral analysis was performed by principal component analysis (PCA) and was conducted with the second derivative spectra between 800\u0026ndash;1800 and 2800\u0026ndash;3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Some spectrum regions were not considered to minimise noise amplification due to derivatives.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Thermogravimetric analysis (TGA)\u003c/h2\u003e \u003cp\u003eThe thermal properties of the lyophilised native discs, treated with collagenase and subjected to the conservation protocols, were studied using a Simultaneous Thermal Analyser, STA 6000 (PerkinElmer, Waltham, MA, USA) under nitrogen with a flow rate of 20 mL/min in the temperature range of 30\u0026ndash;500\u0026deg;C and a heating rate of 10\u0026deg;C/min (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Mechanical testing under compressive stress\u003c/h2\u003e \u003cp\u003eAll samples were subjected to compression tests. A texture analyser was used (TA.XTplusC, Stable Micro Systems, UK), and the assay parameters were: 1.2 mm min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e extension rate, 490 N load cell and the discs were compressed until a strain of at least 55%. The compression modulus was obtained from the elastic region of the graphs (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences between samples were evaluated on GraphPad Prism 8 software. A repeated measurement two-way ANOVA with Sidak\u0026rsquo;s post-hoc test was performed for morphological analysis. A two-way ANOVA with Tukey\u0026rsquo;s post-hoc test was employed for FTIR spectroscopy analysis. For the remaining assays, such as TGA, biochemical quantifications, and mechanical tests, a one-way ANOVA was used with Tukey\u0026rsquo;s post-hoc test. All tests were calculated with a confidence interval of 95%.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Morphological evaluation\u003c/h2\u003e \u003cp\u003eThe native discs presented an anteroposterior dimension of 13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 mm, a mediolateral dimension of 23.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 mm, a thickness of 1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 mm and a weight of 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 g (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When subjected to the degradation and conservation protocols, no statistical differences were found for the anteroposterior length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026ndash; Ai, Aii). For the mediolateral length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026ndash; Bi, Bii), the protocols PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_7 days, Gauze\u0026thinsp;+\u0026thinsp;RT_1 and 14 days led to a decrease of 4.5%, 4.2% and 4.4%, respectively. The thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026ndash; Ci, Cii) was the most affected feature during the degradation process, with a decrease of 37.9%. The PBS\u0026thinsp;+\u0026thinsp;RT_14 days and Gauze\u0026thinsp;+\u0026thinsp;RT_ 7 days protocols also led to thinner discs with a decrease of 11.1% and 17.3%, respectively. Ultimately, for the weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026ndash; Di, Dii), the collagenase treatment led to a decrease of 9.8%. For the gauze\u0026thinsp;+\u0026thinsp;RT protocol, the increase in the freezing time led to a higher percentage of weight loss: 10.9% after 1 day, 14.4% after 7 days and 17.5% after 14 days, in relation to the native disc.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biochemical content evaluation\u003c/h2\u003e \u003cp\u003eThe water, total and soluble collagen, sulphated GAGs, and DNA contents of the native discs were determined for the five regions of the disc. The extent to which disc degradation with collagenase affects the water content and the biochemical composition was also assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe differences in water content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A) between the five regions of the disc were not statistically significant, varying between 72.48 and 77.08%, with the lowest and highest values being attributed to the central and anterior regions, respectively. After degradation with collagenase, the water content increased to 88.28%, with all regions presenting statistically significant differences from the native disc.\u003c/p\u003e \u003cp\u003eTotal collagen content varied from 50.70\u0026ndash;67.71% between regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-B), and soluble collagen varied from 11.09\u0026ndash;13.25% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C), with the anterior and central regions having the lowest content for total and soluble collagen. The opposite was found for sulphated GAGs content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-D), varying from 3.59\u0026ndash;5.35%, with the anterior and central regions having the highest content. When subjected to collagenase, on average, total collagen increased by 3.8%, soluble collagen decreased by 27.4%, and sulphated GAGs decreased by 63.2%. No statistical differences were found for the DNA content between regions, with an average of 0.016% of the disc dry weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the biochemical content as analysed by FTIR spectroscopy, it was observed that the collagenase treatment led to a deviation of the band at 1052 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, assigned to the SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e asymmetric stretching vibrations of sulphated GAGs in the native disc, towards the 1058 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e position (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A, B), and was associated to a statistically significant decrease of sulphated GAGs for the anterior and medial region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-C). No statistical differences were observed between the conservation protocols and the native disc. The conservation protocol, PBS\u0026thinsp;+\u0026thinsp;RT, estimated a content decrease in the posterior region after the 14th day compared to the 1st and 7th days of freeze. For the Gauze\u0026thinsp;+\u0026thinsp;RT protocol, the same behaviour was observed for the anterior region.\u003c/p\u003e \u003cp\u003eBased on the observation of the 1376 cm\u003csup\u003e-1\u003c/sup\u003e band (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-D), which is assigned to CH\u003csub\u003e3\u003c/sub\u003e symmetric bending vibration of sulphated and non-sulphated GAGs, it was estimated a statistically significant increase of sulphated and non-sulphated GAGs content for all regions of the disc. No major statistically significant differences were observed for all the conservation protocols in relation to the native disc. For the conservation protocol PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C, a decrease in sulphated and non-sulphated GAGs content was estimated along the conservation period for the lateral and medial regions between the 1st and 14th days of freeze. No differences were observed along the conservation time for the PBS\u0026thinsp;+\u0026thinsp;RT and Gauze\u0026thinsp;+\u0026thinsp;RT protocols.\u003c/p\u003e \u003cp\u003eBased on the 1338 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-E), which is assigned to CH\u003csub\u003e2\u003c/sub\u003e side chain vibrations of collagen, it was estimated an increase of the collagen content after the collagenase treatment for all discs\u0026rsquo; regions. Compared to the native disc, the conservation protocols resulted in statistically different collagen contents. The protocols leading to a higher decrease of estimated collagen content were the PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_14 days in the central e medial regions and PBS\u0026thinsp;+\u0026thinsp;RT_ 1 day in the central, medial and posterior. Analysing the protocol PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C, a decrease in the collagen content along time was estimated, with higher significance between the 1st and 14th days of freeze for the central, lateral and posterior regions. For the PBS\u0026thinsp;+\u0026thinsp;RT protocol, the opposite estimations were conducted, with a collagen content increase along the conservation period, being more significant between the 1st and 14th days of freeze for all discs\u0026rsquo; regions and between the 7th and 14th days of freeze for lateral and posterior regions. For the Gauze\u0026thinsp;+\u0026thinsp;RT protocols, no statistical differences were estimated along the freezing period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PCA of FTIR spectra shows clusters of scores according to native discs and discs subjected to the collagenase treatment and the different conservation protocols, highlighting their different molecular compositions. Scores associated to PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C and PBS\u0026thinsp;+\u0026thinsp;RT protocols present some overlap in the graphs at the 1st and 7th days of freezing with the scores of the native disc (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e- A, B). Almost no overlap exists between the native disc and scores after 14 days of freezing, when the discs are frozen in PBS, highlighting the high impact of 14 days of freezing on the sample\u0026rsquo;s molecular composition. As for the Gauze\u0026thinsp;+\u0026thinsp;RT protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e- C), after 7 days of freezing, there is a significant impact on the sample\u0026rsquo;s molecular composition in relation to the native disc since no overlap between the scores of these types of samples was observed. The scores from the collagenase-treated disc are the ones further apart in space in relation to all other scores, pointing to a very different biochemical composition in relation to the native disc and in relation to any of the conservation protocols.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Thermal analysis\u003c/h2\u003e \u003cp\u003eThe TGA and the derivative of the TGA (DTGA) show differences between the several experimental groups tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For analysis of the TGA results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the dehydration phase (WL\u003csub\u003e1\u003c/sub\u003e: 30\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;T\u0026thinsp;\u0026lt;\u0026thinsp;100\u0026deg;C), the decomposition phase (WL\u003csub\u003e2\u003c/sub\u003e: 100\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;T\u0026thinsp;\u0026lt;\u0026thinsp;300\u0026deg;C) and the degradation phase (WL\u003csub\u003e3\u003c/sub\u003e: 300\u0026deg;C\u0026thinsp;\u0026lt;\u0026thinsp;T\u0026thinsp;\u0026lt;\u0026thinsp;500\u0026deg;C) were considered. From the DTGA, the denaturation peak of the hydrated collagen (TP\u003csub\u003e1\u003c/sub\u003e), the peak that corresponds to the conformational changes of the collagen molecule from a triple helix structure to the random coil (TP\u003csub\u003e2\u003c/sub\u003e), and the peak that corresponds to the bulk degradation of the collagen fibrils (TP\u003csub\u003e3\u003c/sub\u003e) were also evaluated [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe native disc shows a weight loss of 11.60% in WL\u003csub\u003e1\u003c/sub\u003e, 21.89% in WL\u003csub\u003e2\u003c/sub\u003e and 41% in WL\u003csub\u003e3\u003c/sub\u003e. When degraded with collagenase, it releases a greater amount of water - WL\u003csub\u003e1\u003c/sub\u003e (15.90%) and consequently reduces its weight loss during the degradation phase - WL\u003csub\u003e3\u003c/sub\u003e (35.47%). For the conservation protocols, the samples were mainly affected in the decomposition phase - WL\u003csub\u003e2\u003c/sub\u003e. For the two protocols where the freezing is in PBS, the weight loss percentage is significantly lower on the 1st day, followed by a continuous increase until the 7th day and then until the 14th day, where the values are similar to the native disc. Analysing each protocol individually, this increase in weight loss shows that the 14th day is significantly different from days 1 and 7. The opposite is observed for Gauze\u0026thinsp;+\u0026thinsp;RT, in which on the 1st day of freezing the value is statistically significantly higher than the native disc, but with continued freezing, on days 7 and 14, the value is close to the ones observed for the native disc. This decrease in value also makes days 1 and 14 statistically different.\u003c/p\u003e \u003cp\u003eRegarding the thermal peaks observed in the DTGA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the native disc presented three occurrences: TP\u003csub\u003e1\u003c/sub\u003e at 63.10\u0026deg;C, TP\u003csub\u003e2\u003c/sub\u003e at 237.37\u0026deg;C and TP\u003csub\u003e3\u003c/sub\u003e at 317.20\u0026deg;C. When the disc is degraded with collagenase, both the TP\u003csub\u003e1\u003c/sub\u003e and TP\u003csub\u003e2\u003c/sub\u003e appear at lower temperatures, being significantly different from the native disc. For the conservation protocols, the major differences are found in the TP\u003csub\u003e2\u003c/sub\u003e: the two protocols where the discs were frozen in PBS, during the 1st and 7th day, the peak is non-existent, but at the 14th of freezing, the value is similar to the native disc. As for the Gauze\u0026thinsp;+\u0026thinsp;RT protocol, the peak appears at lower temperatures on the 1st and 7th day. Moreover, in this protocol, the degradation peak - TP\u003csub\u003e3\u003c/sub\u003e occurs at lower temperatures when the disc is frozen for 1 day. Analysing this protocol individually, statistical differences are also found between the different freezing times for TP\u003csub\u003e1\u003c/sub\u003e and TP\u003csub\u003e2\u003c/sub\u003e, in which increasing time leads to an increase in the temperatures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean and standard deviation values of the weight loss between 30\u0026ndash;100\u0026deg;C (WL\u003csub\u003e1\u003c/sub\u003e), 100\u0026ndash;300\u0026deg;C (WL\u003csub\u003e2\u003c/sub\u003e) and 300\u0026ndash;500\u0026deg;C (WL\u003csub\u003e3\u003c/sub\u003e) and the thermal peaks (TP\u003csub\u003e1\u003c/sub\u003e, TP\u003csub\u003e2\u003c/sub\u003e and TP\u003csub\u003e3\u003c/sub\u003e) obtained from the TGA and DTGA analysis for the native, collagenase-treated and subjected to storage discs. Statistically significant differences were calculated with one-way ANOVA with Tukey\u0026rsquo;s post-hoc test and differences represented by * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 are compared with the native disc, and represented by # p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ## p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ### p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 are compared within the same conservation protocol (N\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWL\u003csub\u003e1\u003c/sub\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWL\u003csub\u003e2\u003c/sub\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWL\u003csub\u003e3\u003c/sub\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTP\u003csub\u003e1\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTP\u003csub\u003e2\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTP\u003csub\u003e3\u003c/sub\u003e (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.6 0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e41.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e237.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e318.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollagenase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e35.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e57.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81 **\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e224.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e320.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_1day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 ***\u003csup\u003e, ##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e320.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_7days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 ***\u003csup\u003e, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e64.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e317.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_14days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 \u003csup\u003e##, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e61.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e233.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e315.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;RT_1day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 ***\u003csup\u003e, ##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e39.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e313.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 \u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;RT_7days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 **\u003csup\u003e, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e40.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e317.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePBS\u0026thinsp;+\u0026thinsp;RT_14days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23 \u003csup\u003e##, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e39.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e60.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e230.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e321.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63 \u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGauze\u0026thinsp;+\u0026thinsp;RT_1day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 *\u003csup\u003e, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e59.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24 \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e216.25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46 ***\u003csup\u003e, ##. ###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e310.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98 *\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGauze\u0026thinsp;+\u0026thinsp;RT_7days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e38.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e60.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e226.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 **\u003csup\u003e, ##, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e316.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGauze\u0026thinsp;+\u0026thinsp;RT_14days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e39.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e234.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62. \u003csup\u003e###, #\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e314.52 3.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Mechanical Behaviour under Compressive Loading\u003c/h2\u003e \u003cp\u003eRegarding the mechanical behaviour evaluation, the native disc presents a compressive modulus of 2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 MPa, and after the collagenase treatment, the compression modulus decreased 70% to 0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 MPa. For the PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C protocol, during the 1st and 7th days of freezing, there was a 28% and 32% increase in the modulus, respectively, followed by a stabilisation towards the native disc values at 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-A, D). The opposite was observed for the PBS\u0026thinsp;+\u0026thinsp;RT protocol, in which the modulus remained the same for the 1st and 7th days but increased by 23% after 14 days. For these two protocols, the difference between freezing for 1 or 7 to 14 days is also statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-B, D). Finally, for the Gauze\u0026thinsp;+\u0026thinsp;RT protocol, the modulus increased by 27% after 14 days of frozen (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe characterisation of animal-derived tissues is extremely important to assess their potential to regenerate or replace human tissues. Different studies have evaluated different animal models and reported that the ovine model is suitable for TMJ research [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, detailed information on its DNA, water and biochemical contents, particularly in terms of its soluble collagen, and a subsequent comparison with a degraded disc has not yet been evaluated. Furthermore, an optimal conservation protocol is yet to be established.\u003c/p\u003e \u003cp\u003eIn the current work, it was observed average values for native ovine TMJ disc were 75.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% water, 59.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4% total collagen, 11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% soluble collagen, 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% sulphated GAGs, and 0.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002% DNA by dry weight. These values are similar to the human TMJ discs [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Interestingly, it was also found that the anterior and central regions presented the lowest collagen content and the highest sulphated GAGs content for the ovine TMJ disc. The remaining regions display the inverse correlation. Interestingly, this inverse correlation between the amount of sulphated GAGs and type II collagen is usually found in the different zones of articular cartilage [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollagenase treatment has a detrimental effect on the collagen network by cleaving the chains of the triple helix into small fragments and enabling the subsequent action of other enzymes, such as gelatinases [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These fragmentations have been observed in internal derangements of the human disc [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], so the use of this enzyme was important to assess the extent to which the conservation protocols damage the TMJ disc. The degraded native disc turned into a gel-like structure, which consequently led to a decrease in thickness and weight. In addition, as expected, the water content increased significantly since in osteoarthritic samples this also occurs due to the breakdown of the collagen network [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The total and soluble collagen contents increased and decreased, respectively. Despite the increase in total collagen, this was only found for the anterior and central regions. This may be due to the portion of the disc analysed since, after freeze-drying, the disc remained in a paper-like structure, making it impossible to distinguish and divide the five regions. So, in practice, collagenase treatment can lead to a stabilisation or increase in total collagen. This phenomenon is attributed to the limited efficacy of collagenases in cleaving insoluble collagen, which is characterised by a higher degree of crosslinked fibres [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Interestingly, it has also been reported that subjecting insoluble collagen type II to gastric pepsin results in an elevated degree of crosslinked aggregation within the collagen fibres, while for soluble collagen, the fibres almost disappear [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It should be noted that collagenase treatment, by leading to the increase of total collagen, may negatively affect cellular behaviour during tissue remodelling by leading to an increase of total collagen [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Collagenase treatment also led to a significant decrease of sulphated GAGs, most probably due to the disintegration of the collagen network, resulting in GAGs release [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This is concordant with Fazaeli et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] observation of a decrease in sulphated GAGs despite not finding a statistically significant reduction in collagen. The authors attributed this low reduction to the time and concentration of the enzyme. Compared to the present study, the animal model may also have contributed to this difference, as the ovine model presents a smaller disc when compared to the porcine model [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], resulting in a much more effective action of the enzyme. Ultimately, all these changes in biochemical content led to a drastic reduction in the compressive modulus, which is in accordance with the reported for the porcine TMJ disc [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese changes in the collagen structure were further validated by TGA and DTGA analysis, where \u003cem\u003e(i)\u003c/em\u003e a greater amount of water was released (WL\u003csub\u003e1\u003c/sub\u003e), \u003cem\u003e(ii)\u003c/em\u003e the weight loss in the degradation zone (WL\u003csub\u003e3\u003c/sub\u003e) was significantly lower, and \u003cem\u003e(iii)\u003c/em\u003e the peak corresponding to changes in the collagen triple helix (TP\u003csub\u003e2\u003c/sub\u003e) also appeared significantly earlier.\u003c/p\u003e \u003cp\u003eTo evaluate the ECM of a cartilaginous tissue, usually diverse laborious and complex methods are conducted based on histology and biochemical testing [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. FTIR spectroscopic analysis may present complementary information concerning the sample molecular characterisation while enabling a simple, rapid (a spectrum is typically acquired in 1 min) and economic (no expensive reagents are required) analysis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The spectrum represents vibration modes of diverse functional groups and, consequently, may be used to evaluate the sample's whole molecular composition and estimate the quantity of diverse biomolecules [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Despite the advantages of the FTIR spectroscopy technique, it can present low specificity due to overlapping bands and even due to common bonds present in different molecules [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Spectra derivatives may enable the deconvolution of some overlapping bands, increasing the spectra resolution [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Indeed, diverse authors have used the FTIR technique to estimate collagen and GAGs in articular cartilage of steers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], humans and bovines [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the analysis of the second derivative spectra, it was estimated that the collagenase treatment resulted in a decrease in the content of sulphated GAGs, with an average decrease of 62.9% for all regions when compared to the native disc, which is in accordance with the results obtained with the staining methods (63.2%). This decrease was also validated by the shift of the band to a higher wavelength (1052 to 1058 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), as previously shown to occur when elastin content decreases [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. For collagen, a 47.6% increase was inferred based on spectra analysis. A small part of this increase is in accordance with the 3.8% increase of total collagen as determined by the staining methods. However, most probably, based on the FTIR spectra, most of the increase results from the higher exposition of the aminoacids due to collagen fragmentation by collagenase. Thus, the increased infra-red absorption of collagen is mainly due to a significant structural and conformation change of collagen and not due to an increased quantity of collagen. This is concordant with the observations based on polarised light microscopy of the loss of structural integrity after collagenase treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. There was also an increase in content for all GAGs (sulphated and non-sulphated). Although the band at 1376 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is generally used to quantify all GAGs, it also represents the absorption of glycoproteins [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, this absorption increase can also result from the significant increase absorption of glycoproteins, due to proteins' significant structural changes. Spectral PCA also reinforces this by pointing a significant alteration of the molecular composition between the native and the collagenase-treated discs.\u003c/p\u003e \u003cp\u003eRegarding the conservation protocols, the one that most affected the morphology was the Gauze\u0026thinsp;+\u0026thinsp;RT, which led to a more dehydrated disc and, consequently, to its reduction in thickness and weight. Based on the ECM analysis by the second derivative, the discs composition was not significantly affected by the conservation protocols in comparison to native discs. However, some impact on the disc\u0026rsquo;s composition was observed in the spectra PCA, although it was much smaller than the observed with the collagenase treatment.\u003c/p\u003e \u003cp\u003eWith TGA and DTGA, it was possible to observe the impact on the collagen structure. Both protocols in which the discs were frozen in PBS present similar results, where for days 1 and 7 the weight loss in the decomposition phase (WL\u003csub\u003e2\u003c/sub\u003e) was lower, and the decomposition peak (TP\u003csub\u003e2\u003c/sub\u003e) was non-existent. Gelatine, which is composed of denatured collagen, presents the collagen fibres in random coil, meaning that the TP\u003csub\u003e2\u003c/sub\u003e peak was also not found [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, freeze and thawing caused this modification in collagen fibres. Contradictorily, for the Gauze\u0026thinsp;+\u0026thinsp;RT protocol, WL\u003csub\u003e2\u003c/sub\u003e presented a significantly higher weight loss on day 1, and for TP\u003csub\u003e2\u003c/sub\u003e the peak for days 1 and 7 appears at a lower temperature due to lower thermal stability, as reported to occur in tendons [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and rat tail collagen-based hydrogels [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] upon freeze and thawing. Interestingly, day 14 showed a stabilisation of the collagen network for all protocols by presenting results similar to the native disc. It is known that protein breakdown is caused by the formation of ice crystals [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], which ultimately appear to have a greater impact on the 1st and 7th day of freezing.\u003c/p\u003e \u003cp\u003eIt was also observed by FTIR spectroscopy, on all conservation protocols, a significant impact along the freezing period. To better analyse this, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-A shows the average values for all protocols, simultaneously considering all disc\u0026rsquo;s regions. It was possible to observe a pattern for sulphated and non-sulphated GAGs (1376 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and for collagen (1338 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): for the PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C protocol, increasing freezing times leads to a decrease of these compounds, whereas for the PBS\u0026thinsp;+\u0026thinsp;RT protocol, an increase of these compounds was found. Regarding sulphated GAGs (1052 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) a decrease was observed for all conservation protocols as the freezing time increased.\u003c/p\u003e \u003cp\u003eMechanically, the conservation protocols also had a detrimental effect on the compressive performance of the discs. PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C led to a stiffer disc on the 1st and 7th of freezing, while PBS\u0026thinsp;+\u0026thinsp;RT was on the 14th day. For Gauze\u0026thinsp;+\u0026thinsp;RT, Allen \u0026amp; Athanasiou [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] also investigated this protocol and reported that the discs can be frozen up to 5 times without altering their viscoelastic properties. However, with the same protocol but with NaCl instead of PBS, Calvo-Gallego et al. showed that the viscoelastic properties change after 30 days of freezing [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although in both studies only the intermediate/central zone was analysed, and the animal model chosen was the pig, these results are in agreement with those found in the present study, as with this protocol, there were alterations in the compression capability after 14 days frozen. This alteration was defined to be due to the loss of interstitial fluid in the discs, since a more dehydrated disc was found.\u003c/p\u003e \u003cp\u003eThe mechanical performance of the TMJ disc is defined by the cooperation of the different ECM components, and it is characterised as a viscoelastic structure, as it helps to absorb stress and distribute loads on the disc, cartilage and bone components [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In order to find out if there is a relationship between the quantitative results from the FTIR bands and the mechanical behaviour, three graphs were plotted, in which only the average is presented for better visualisation. In Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-B, presents the relationship between sulphated GAGs (band 1052 cm-1) and the compression modulus. It was observed that both parameters of collagenase-treated and Gauze\u0026thinsp;+\u0026thinsp;RT_1 days decreased, while for the PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_14 days and PBS\u0026thinsp;+\u0026thinsp;RT_1 days, they increased. However, for the remaining protocols, the decrease of sulphated GAGs content led to an increase in the stiffness of the disc. Regarding the sulphated and non-sulphated GAGs content (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-C), it was observed that with the increase of the biochemical content, there is also an increase in the compression modulus. The same is found in the relationship with the collagen content (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-D). The protocols that such pattern is not extrapolated is the PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C_7 days for sulphated GAGs and PBS\u0026thinsp;+\u0026thinsp;RT_7 days for sulphated GAGs and collagen. Regarding the discs treated with collagenase, this pattern is also not found because, as explained above, the increase in biochemical content for collagenase-treated samples in the FTIR analysis is actually indicative of its fragmentation. Furthermore, through all the characterisations reported in this study, no profile similar to collagenase-treated samples was found in the discs subjected to the protocols.\u003c/p\u003e \u003cp\u003eAlthough it has been reported that the collagen content is related to the tensile capacity of the TMJ discs, Detamore et al. highlighted the challenge of establishing a correlation between the content of sulphated GAGs and the compressive performance [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Furthermore, Willard et al. pointed that even after removing 96% of the sulphated GAGs using chondroitinase, there was no significant change in the instantaneous compressive modulus [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. On the other hand, collagen appears to exert a substantial influence on the compressive capacity of the TMJ discs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. These results agree with those found in the present study, in which was found a stronger relationship between compression tests and collagen content when compared to sulphated GAG. For total GAGs, there also seems to be a high association. However, as referred above, this band seems to have influence from glycoproteins, so extrapolating that, the mechanical behaviour is related to total GAGs is more complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, for the discs frozen in PBS, it was possible to conclude that the type of thawing can result in opposite behaviours as the results of the compression tests align with those obtained for the quantification of the biochemical content. However, regarding the morphological and thermal properties, the impact of the thawing method does not appear significant as similar results were obtained. Conversely, when the thawing process is the same, different results are found across all evaluated parameters, demonstrating once again the influence of the freezing method on the disc's native characteristics. Our results suggest that all the protocols induced alterations in the native properties of the ovine disc, so in the case of using the full disc for TE strategies, these should be taken into account. However, if the disc is to be used in powder form, for example, to develop minimally invasive strategies, then we recommend storing the discs in Gauze\u0026thinsp;+\u0026thinsp;RT for 14 days, as the dehydration and mechanical property changes observed by the 14th day become negligible.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by the Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e a Tecnologia (FCT) for its financial support through the following projects from CDRSP: UIDB/04044/2020, UIDP/04044/2020, Associate Laboratory ARISE (LA/P/0112/2020), InnovaBIOMAS (2022.10564.PTDC); from IBB: UIDB/04565/2020, UIDP/04565/2020, and Associate Laboratory i4HB (LA/P/0140/2020); and PhD studentship: 2022.12030.BD. This research was also funded through the institutional scientific employment program-contract (CEECINST/00077/2021).\u003c/p\u003e\u003ch2\u003eAuthors' contributions:\u003c/h2\u003e \u003cp\u003eConceptualization: DT, AM, NA, CM; Methodology: DT, AM, NA, CM; formal analysis: DT, CC, JS; Writing and revising of the article: DT, CC, JS, AM, NA, CM. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDonahue RP, Hu JC, Athanasiou KA (2019) Remaining Hurdles for Tissue-Engineering the Temporomandibular Joint Disc. 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Arch Oral Biol 86:1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Temporomandibular joint disc, Ovine model, Biochemical composition, Freezing time storage, Extracellular matrix","lastPublishedDoi":"10.21203/rs.3.rs-4964539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4964539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe temporomandibular joint disc plays a fundamental role in daily activities, and when it is compromised, severely disturbs oral health and quality of life. Decellularization is gaining interest in tissue engineering (TE) applications, but requires maintaining the native structure and composition to mimic human disc properties. This study characterizes the native ovine disc and compares conservation protocols to preserve its morphology, biochemical content (sulfated glycosaminoglycans, total and soluble collagen), and mechanical and thermal behavior. Three storage protocols were tested: \u003cem\u003e(i)\u003c/em\u003e freezing at -20\u0026deg;C in phosphate-buffered saline (PBS) and thawing at 4\u0026deg;C (PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C); \u003cem\u003e(ii)\u003c/em\u003e freezing at -20\u0026deg;C in PBS and thawing at room temperature (RT) (PBS\u0026thinsp;+\u0026thinsp;RT); and \u003cem\u003e(iii)\u003c/em\u003e wrapping the discs in PBS-embedded gauze, freezing at -20\u0026deg;C, and thawing at RT (Gauze\u0026thinsp;+\u0026thinsp;RT). Protocols were evaluated at 1, 7, and 14 days, and compared with a native disc, and a collagenase-treated discs. All conservation protocols caused changes, though less pronounced than degradation. The PBS\u0026thinsp;+\u0026thinsp;4\u0026deg;C and PBS\u0026thinsp;+\u0026thinsp;RT protocols maintained original morphology, yet highlighted, a contrasting biochemical and mechanical outcome based on the thawing method. Thermal analysis revealed collagen structure changes within the first 7 days of freezing. The Gauze\u0026thinsp;+\u0026thinsp;RT protocol showed no significant biochemical changes over time, but the disc became dehydrated and with a higher compression modulus. For TE approaches involving decellularization, it is crucial to consider these alterations. For powdered tissue applications, the Gauze\u0026thinsp;+\u0026thinsp;RT method for 14 days is recommended due to minimal structural impact.\u003c/p\u003e","manuscriptTitle":"Biochemical and Mechanical Impact of Storage Techniques on Ovine Temporomandibular Joint Discs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 10:00:59","doi":"10.21203/rs.3.rs-4964539/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f0fd4190-3bbb-44ef-b196-9c69e1092ba2","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T10:00:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-26 10:00:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4964539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4964539","identity":"rs-4964539","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}