The use of antimicrobial growth promoters (AGPs) in animal production has been associated with the increase in bacteria resistance to multiple drugs. The ban on the use of AGPs in many countries has highlighted the increasing need for alternatives as non-antibiotic growth promoter feed additives. Thus, a study was conducted on broiler chickens to test the effectiveness of sodium salicylate  as a non-antibiotic anti-inflammatory agent. The results showed that sodium salicylate did not have a significant effect on the health of animals; however, the results suggest that further studies may be necessary under more stressful conditions to better evaluate the efficacy of these agents as growth promoters.Antimicrobial growth promoters (AGPs) in animal production have been related to the increase in multidrug-resistant bacteria. The AGP ban in many countries has highlighted the growing need for alternatives for feed additives. Considering the non-antibiotic anti-inflammatory theory of AGPs, chicks received three different doses of sodium salicylate (SS) in feed , basal diet (BD) was used as a negative control, and zinc bacitracin (ZB) was used as a positive control. Chicks were individually housed to increase the accuracy of the dose of SS ingested. Performance parameters and footpad dermatitis were evaluated weekly, while haematology, serum biochemistry, histopathology, and tibial dyschondroplasia were determined on Days 21 and 42. A linear dose-dependent decrease in haemoglobin concentration was observed, but the values were within the normal reference range. Among all the other evaluated parameters, no relevant differences between treatments were observed; however, not even the AGP group performed better than the control group. It is possible that the conditions in which the birds were raised were not stressful enough to allow for anti-inflammatories to demonstrate their beneficial effects on performance. Studies should be conducted where the animals are exposed to commercial conditions, as the presence of natural stressors could allow a better evaluation of the efficacy of the anti-inflammatory agent as a growth promoter. Poultry is the main animal protein source for human nutrition, with a global production of more than 100 million tons in 2021 . This feBanning AGPs without replacement options will result in reduced animal performance and an increased incidence of animal disease , which aTo develop effective non-antibiotic alternatives, it is first necessary to understand the biological mechanism of action of AGPs. There are two main theories, one of which posits that AGPs have antibacterial action on the intestinal microbiota, reducing subclinical infections and improving animal performance ,10. The In birds, non-steroidal anti-inflammatory drugs, especially sodium salicylate (SS) and acetylsalicylic acid (ASA), are frequently used due to their anti-inflammatory and analgesic properties . These cThus, we hypothesized that the prolonged use of SS would improve the growth performance of broiler chickens by decreasing the inflammatory process in the gastrointestinal tract. To test this hypothesis, in the present study, we aimed to evaluate the safety of SS individually applied during all cycles of the production of broiler chickens.The sodium salicylate (SS) used was grade PA . An assay was carried out to determine the saturation solubility coefficient (shake-flask method) of SS in ethanol, methanol, and acetonitrile. For this, SS, in sufficient quantity to saturate the system, was added to 20 mL of solvent and stirred for 24 h. Afterwards, an aliquot of the supernatant was filtered in PTFE, hydrophilic 0.22 µm, and diluted 1:100, and the reading was performed using UV spectrophotometry using the 297 nm wavelength filter . Assays were performed in triplicate.The incorporation of the drug was carried out via cornmeal, adding the drug and water until the final concentration was 5.12% SS. The high viscosity conferred on the wet mass by the SS prevented its inclusion at higher concentrations. Afterwards, the granules were manually prepared using a 1.18 mm sieve and kept in a forced circulation oven  at 45 °C for 7 h. Residual moisture was monitored on an electronic moisture scale , where 0.5 g of the granulate was evaluated in triplicate, and the process ended when the residual moisture was equal to or less than 5%.2 = 0.9948.The calibration curve was obtained through serial dilution in methanol of the SS standard containing 62.70 µg/mL in 6 levels  in triplicate. The reading was performed by UV spectrophotometry , obtaining RBriefly, the extraction procedure was performed by adding 50 mL of methanol to 20 mg of granules, vortexed for 30 s, sonicated for 15 min, and left to rest for 2 h. Subsequently, the solution was again vortexed, sonicated, and centrifuged at 5000 RPM for 10 min (20 °C). The supernatant was analysed by UV spectrophotometry. The recovery of SS was 106.7%, and the coefficient of variation was 1.1%.A total of 70 one-day-old Cobb 500 male broiler chicks vaccinated against Marek and Gumboro diseases were purchased from a commercial hatchery and were used. The experiment was performed in the experimental houses of the Poultry Research Laboratory of the Department of Animal Nutrition and Production .Until the 21st day of life, chicks were individually housed in stainless steel vertical batteries  equipped with trough-type feeders, nipple drinkers, and concealers. On the 22nd day, the birds were transferred to galvanized steel wire cages  equipped with nipple drinkers and with controlled temperature and photoperiod. Animals were maintained at a comfortable temperature according to their age, with feed and water ad libitum, and a controlled photoperiod.Diets were based on corn and soybean meal formulated for a two-phase feeding program  to meet the nutritional and energy recommendations suggested by Rostagno et al.  Table 1Table 1.n = 14 per group): (1) negative control, which received only the basal diet (BD group); (2) positive control, which received the basal diet supplemented with 55 ppm zinc bacitracin (ZB group); and three groups, which received the basal diet supplemented with SS granules (5.12%) at doses of 10, 30, or 90 mg of sodium salicylate per day . The doses used in this experiment were based on findings from a previous experiment [Broilers were randomly allocated into five treatment groups  and body weight (BW) were assessed weekly, and the performance variables body weight gain (BWG) and feed conversion ratio (FCR) were calculated for each period. The broilers were measured individually.Blood samples were collected at the 21st and 42nd day from 12 and 9 birds per treatment, respectively, by ulnar venipuncture. Blood samples were placed in K3 EDTA and serum separator clot activator plastic tubes  for haematological and biochemical analysis.g for 5 min, and the results were estimated as a percentage. Determination of haemoglobin concentration was performed by the cyanomethemoglobin method using the reagent from Bioclin®  and evaluated using a spectrophotometer . Mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and mean corpuscular volume (MCV) were calculated.The haematological evaluation was performed by a manual method in a Neubauer chamber using blood diluted on 0.01% toluidine blue stain at a 1:100 dilution. Differential white blood cell counts were made using an average of 100 cell counts from blood smears with Wright’s stain and examined under an optical microscope with a 100X objective. Haematocrit was determined in whole blood using a micro-haematocrit tube centrifuged at 11,500× ®  according to the manufacturer’s instructions. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma glutamyl transferase (GGT), total protein (TP), albumin (ALB), creatine kinase (CK), glucose (GLU), creatinine (CR), and uric acid (UA) were evaluated.Serum biochemistry was analysed using an automated biochemical analyser  with BioclinOn the 21st and 42nd days, 5 and 6 broilers from each group, respectively, were euthanized, and samples from the crop, proventriculus, gizzard, small intestine , caecum, liver, and kidneys were collected for histopathological evaluation. The samples were fixed in 10% buffered formaldehyde and processed according to routine protocols. Sections (5 µm) were stained with haematoxylin and eosin and examined under an optical microscope. Lesions were classified as mild, moderate, or severe according to their intensity and as focal, multifocal, or diffuse based on their distribution.https://imagej.net/Fiji/Downloads, accessed on 1 March 2023). The growth cartilage thickening of these bones was evaluated, and scores were assigned according to the degree of injury: 0, no evidence of TD; 1, thickening between 1 and 3 mm; 2, thickening between 3.1 and 6.0 mm; and 3, thickening greater than 6 mm [The legs of the chickens were separated during slaughter and frozen at −20 °C until processing and analysis of tibial dyschondroplasia (TD). The macroscopic evaluation was performed on the right tibia samples, which were stripped and cut longitudinally. Measurements were performed using the ImageJ program . Statistical analysis was performed with SAS (version OnDemand for Academics) and analysed using the Generalized Linear Mixed Models procedure with the alpha value set at 0.05. Linear and quadratic polynomial contrasts were specified for dose–response modelling with correction for unequal spacing of SS doses, namely, 0, 10, 30, and 90 mg/kg per day. Dunnett’s multiple comparison test was used to compare the reference diet (zinc bacitracin group) against the basal diet and diets formulated using SS. Data subjects with repeated measurements from the same individuals over a period of time were subjected to the linear mixed model, with treatment, time, and the treatment x time interaction as fixed. In the case of multinomial (ordered) response distributions , the cumulative logit function was used to compare all treatments. The possible existing correlations between the studied variables were verified through CORR procedure of SAS. p = 0.0397). ZB also promoted an increase in BW at week 5 compared to the BD (0) and S10 groups (p = 0.0131). In the first week, the feed intake and feed:gain ratio presented a quadratic response . ZB also increased feed intake and the feed:gain ratio compared to the BD group . At week 4, ZB again reduced feed intake compared to the BD group (p = 0.0005). The addition of SS promoted a linear reduction in consumption in the sixth week (p = 0.0147), but it was not sufficient to impact the periods from 22 to 42 days and the total period of the experiment. There was a significant interaction between time and treatment (p ≤ 0.05) observed for FI.The effects of the five dietary treatments on BW, FI, and F:G are summarized in p = 0.0158) and 42 days (p = 0.0006) and a significant difference between ZB and S90 . This resulted in a significant linear reduction in MCH and MCHC on Day 42  and a lower value for MCH and MCHC in the S90 group compared to ZB on Day 21 (p = 0.0327 and p = 0.0253) and MCHC on Day 42 (p ≤ 0.0001). MCH also presented a significant interaction between age and treatment (p = 0.0462). Regarding the leukogram, only the HL ratio showed a significant linear reduction on Day 21 (p = 0.0272). All other parameters did not show significant linear or quadratic differences in relation to the reference group (ZB) or even the age–treatment interaction.The haemogram  revealedp = 0.0499). At this age, the use of ZB was also able to significantly reduce LDH compared to the BD group (p = 0.0384). At 42 days, significant linear decreases in ALT (p = 0.0472) and AST (p = 0.0111) and significant linear increases in TP (p = 0.0019), GLOB (p = 0.0022) and GLU (p = 0.0142) were observed. On the 42nd day, ZB significantly affected TP compared to the BD group (p = 0.0024) and GLU compared to the S90 group (p = 0.0220). Only ALP presented a significant interaction between age and treatment (p = 0.0106).In serum biochemistry  on Day 2p = 0.0350), WG , and F:G ratio  of the birds; however, these correlation patterns were not significant at 42 days.In the histopathological evaluation of the small and large intestines, liver, and kidney, the presence of mild to moderate inflammatory infiltrate was verified in the groups treated with SS and in the BD and ZB groups; however, none of the changes showed a significant difference between treatments (data not shown). Although the histopathological evaluation showed no significant difference between the treatments, at 21 days, there was an increase in the average scores of renal mononuclear infiltrates according to the increase in the SS dose. This finding had a significant correlation with the BW , such as erosive damage or upper gastrointestinal bleeding, were observed in birds from any of the treatment groups.None of the animals across all treatment groups exhibited any lesions associated with pododermatitis or tibial dyschondroplasia (data not shown).The gastrointestinal tract has the difficult job of striking a balance between being an effective barrier and being selectively permeable to nutrients and tolerant of the associated resident microbiota . IntestiOne of the main and most accepted theories about the growth-promoting effect of antimicrobial drugs is around their non-antibiotic anti-inflammatory effects ,12. ReinA search in the literature to provide data about the beneficial use of ASA supplementation in the diets of birds under different stress conditions ,33,34,35While the pharmacokinetics and pharmacodynamics of ASA and SS and their capacity to treat inflammation in birds are well known ,42,43, dIn relation to the biochemical assessment, in the same manner as a previous study conducted by our group employing lower doses of SS  in broilers for 42 days, we did not detect consistent differences in serum parameters evaluated .Concerning histopathological evaluations, although studies have shown that ASA promotes lesions in the small intestine ,47, lungEnvironmental stresses, such as inadequate nutrition, crowding, moving and mixing of animals, poor sanitation, and high or low temperatures, contribute to increased responses to AGPs ,50,51. TIn the present study, we did not detect significant alterations in growth performance between the different groups at the end of the treatment. Thus, it is possible to suppose that in the same manner as AGP, anti-inflammatory drugs could show the greatest effects under stressful conditions. In fact, no changes were observed even in the animals of the positive control group, ZB, where an increase in weight gain would be expected since it is well established that zinc bacitracin improves growth performance . This hyTibial dyschondroplasia and footpad dermatitis are pathological conditions commonly faced in commercial poultry and are associated with many factors, and both diseases reduce overall welfare, technical performance, and carcass yield in broiler chickens ,56. SincGathering data from these toxicological evaluations, we can assume that doses of SS up to 90 mg/kg administered daily for 42 days caused a reduction in haemoglobin concentration as the only adverse effect. Nevertheless, studies are needed to verify the relevance of this finding. Moreover, sodium salicylate supplementation was not significantly efficient in promoting performance improvement; however, not even the birds treated with ZB showed improvement in performance when compared to those of the control group. In this way, it is possible that the conditions under which the birds were raised were not stressful enough to allow for the possible positive effects of both SS and ZB to be highlighted as growth promoters. Thus, studies should be conducted in which the animals are exposed to commercial conditions, where natural stressors could better evaluate the anti-inflammatory efficacy on performance.


This research underscores the potential of integrating HSI technology with DL methods, thereby establishing the feasibility of HSI as an efficient and non-destructive tool for predicting the content of isoflavones and starch in PTR. Moreover, this methodology holds great promise for enhancing efficiency in quality control within the food industry.Accurate assessment of isoflavone and starch content in Puerariae Thomsonii Radix (PTR) is crucial for ensuring its quality. However, conventional measurement methods often suffer from time-consuming and labor-intensive procedures. In this study, we propose an innovative and efficient approach that harnesses hyperspectral imaging (HSI) technology and deep learning (DL) to predict the content of isoflavones  and starch in PTR. Specifically, we develop a one-dimensional convolutional neural network (1DCNN) model and compare its predictive performance with traditional methods, including partial least squares regression (PLSR), support vector regression (SVR), and CatBoost. To optimize the prediction process, we employ various spectral preprocessing techniques and wavelength selection algorithms. Experimental results unequivocally demonstrate the superior performance of the DL model, achieving exceptional performance with mean coefficient of determination (R It is vAlongside its medicinal value, PTR is extensively acknowledged as a nutritious food, providing essential nutrients such as starch, protein, and fiber that are integral to maintaining a balanced and healthy diet . RemarkaWith the continuous improvement of living standards in the modern era, consumer concerns regarding food quality have gained significant prominence. Safeguarding the commercial value of PTR necessitates a focus on controlling the content of its bioactive compounds to ensure quality attributes . To faciNear-Infrared Spectroscopy (NIR) and Infrared Spectroscopy (IR) techniques offer notable advantages in substance analysis , includiDeep learning (DL) has gained tremendous popularity in recent years, driven by its remarkable ability to tackle complex problems . aging, As mentioned above, the combination of HSI and DL algorithms have shown significant potential for application in the field of food analysis in recent years. In this context, the main objectives of this study are as follows: (1) Explore the potential of HSI and data analysis methods to predict the levels of puerarin, puerarin apioside, daidzin, daidzein, and starch in PTR. (2) Following the conventional modeling process, compare the performance of typically used traditional machine learning prediction algorithms, with various spectral preprocessing techniques and wavelength selection methods. (3) Develop a 1DCNN model and establish its advantage by comparing its predictive performance with commonly used prediction algorithms. By addressing these objectives, this study aims to advance the accurate prediction of compounds in PTR using HSI technology and data analysis methods, providing valuable insights for quality evaluation and control in the food industry.22.1Pueraria thomsonii) sourced from Jiangxi Province, China. The kudzu plants were harvested during their second year of growth, and the root tuber epidermis was carefully removed using ultrasound-assisted washing. The PTR samples were then subjected to natural drying. A total of 1000 g of PTR was collected from the specified geographical origin, and 10 g of PTR were packed together to form one sample for hyperspectral image acquisition. In total, 100 samples were collected for analysis. During hyperspectral image acquisition, the PTR samples were individually placed on a black plate. Subsequently, the samples were freeze-dried and finely ground into powders to facilitate the analysis of puerarin, puerarin apioside, daidzin, daidzein, and starch.This study focused on kudzu . Specifically, our focus for the experiment was on the wavelength range of 948.72 − 2512.97 nm in the SWIR region, which comprised a total of 288 spectral bands. To ensure proper illumination of the samples, we employed two 150 W bromine-tungsten lamps  as the light source. The integration time for the SWIR lenses was set at 3500 2.3During the process of acquiring hyperspectral images, non-uniformity in the intensity of light and interference from dark currents can lead to uneven output images . This canewI, and it is obtained by applying a correction method to the original hyperspectral image rawI, taking into account the dark reference image darkI and the white reference image whiteI.The corrected image is denoted by 2.42.4.1Methanol (chromatography grade) and acetonitrile (chromatography grade) were obtained from Tianjin Siyou Fine Chemical Co., Ltd. Methanol  and formic acid  were obtained from Tianjin Zhiyuan Chemical Reagent Co., Ltd. Ultra-pure water was prepared in the laboratory using a Mili-Q Advantage A10 system from Merck KGaA . The standards used in this study included puerarin (lot number 110752-201816), daidzin (111738-201904), daidzein (111502-202003), which were purchased from the China Institute for Food and Drug Control. Furthermore, puerarin apioside (lot number 103654-50-8) was purchased from Chengdu Plantmark Pure Biotechnology Co., Ltd. All reagents were of analytical grade and were used without further purification.2.4.2Four distinct compounds, namely puerarin, puerarin apioside, daidzin, and daidzein, were each dissolved in methanol to prepare standard solutions with a 20 mg amount of each compound. The solutions were made up to a final volume of 10 mL in volumetric flasks to obtain the stock solutions for each compound. To prepare a mixed standard solution, suitable volumes of the stock solutions were combined and diluted with methanol to produce a concentration gradient. Standard curves were created for each compound using the appropriate volumes of the standard solutions. This rigorous method was employed to guarantee precise and dependable measurements of the compounds in subsequent experiments. The resulting mixed standard solution and individual standard curves will be used to determine the concentration of each compound in samples.2.4.3µm microfiltration membrane and then injected into a sample bottle using an automatic sampler.Firstly, 1.0 g of the powder, which had passed through a sieve (50 mesh), was mixed with 50 mL of the 50% methanol solution. Ultrasonic extraction was then performed on the mixture at room temperature for 30 mins. The resulting mixture was cooled, and its weight was measured. Any weight loss was compensated for by adding more 50% methanol solution to the mixture. After shaking, the mixture was filtered, and the filtrate was collected for further analysis. To prepare the filtrate for injection, it was filtered through a 0.22 2.4.4The HPLC K2025 liquid chromatography system was composed of a Binary pump, Autosampler, Column oven, and UV-VIS detector. For every analysis of the samples, a YMC-Pack Pro C18 reverse phase column was utilized with a mobile phase consisting of an aqueous solution containing 0.01% formic acid and acetonitrile. Detection was conducted at a wavelength of 250 nm, with the column temperature maintained at 30 °C, a flow rate of 1 mL/min, and an injection volume of 10L.2.4.5A sample of PTR weighing precisely 1.0 g, which had been passed through a sieve (50 mesh), was added to 10 mL of distilled water. The resulting mixture was homogenized in a blender, filtered, and the filtrate was subjected to centrifugation at 4500 rpm for 15 mins. The supernatant was then discarded, and the resulting precipitate was dried in a 70 °C oven until a constant weight was achieved. The mass of the precipitate was recorded for the calculation of starch content using the following formula.m1 is the mass of the dried precipitate, and m2 is sample mass.Where, W is starch content, 2.52.5.1Preprocessing plays a crucial role in hyperspectral data analysis as it helps eliminate noise, correct instrument artifacts, and enhance the signal-to-noise ratio for further analysis. In this study, we evaluated several well-established preprocessing methods, including the standard normal variate (SNV), multiplicative scatter correction (MSC), Savitzky-Golay smoothing (SG), as well as the first derivative (FD) and second derivative (SD) methods. The goal was to identify the most effective preprocessing technique that would yield the most accurate predictive models.2.5.2In the analysis of hyperspectral data, the selection of appropriate spectral bands is of paramount importance for obtaining meaningful information and reducing data redundancy. Given the hundreds or thousands of spectral bands, noise and irrelevant spectral information are common, which can lead to inaccurate results or high computational costs in further data analysis. Effective band selection aims to improve the accuracy and efficiency of the analysis by reducing the dimensionality of the data while preserving the most informative spectral features. Furthermore, the selection of relevant bands can facilitate the interpretation of the data and provide insights into the underlying physical and chemical processes. Hence, it is a critical step in hyperspectral data analysis and has received significant attention in the literature.Successive projections algorithm (SPA): The SPA operates by computing the coefficient of determination between each spectral band and the target variable, and subsequently selecting the feature with the highest absolute correlation in the initial iteration (teration . During Competitive adaptive reweighted sampling (CARS): CARS is a potent algorithm that facilitates wavelength selection in HSI. Its underlying principle involves competitively sampling spectral bands iteratively. Every wavelength is assigned a weight that corresponds to its significance in the classification task (ion task . CARS adUninformative variable elimination (UVE): This algorithm achieved by measuring the relevance of each wavelength to the final task using statistical methods such as mutual information, coefficient of determination, or variance analysis. Wavelengths that exhibit low significance are subsequently removed, resulting in only informative wavelengths remaining for subsequent analysis : SVR is a powerful machine learning algorithm that is extensively used for regression tasks. The fundamental principle of SVR is to construct a hyperplane in a high-dimensional feature space that maximizes the margin between the training data points and the hyperplane, facilitating accurate regression : In the realm of HSI, PLSR is a frequently employed multivariate statistical technique for predicting the concentrations of chemical components in a sample. Its primary objective is to reveal the underlying relationship between the predictor variables  and the response variable .PLSR achieves this by decomposing the predictor variable set into a reduced number of latent variables that capture the variance in the spectral data. It then performs regression analysis between these latent variables and the response variable. The latent variables are carefully selected to maximize the covariance between the predictor and response variables, thereby ensuring that they capture the critical information and relationships between the two sets .CatBoost: Through the use of ordered boosting, random permutations, and gradient-based optimization, CatBoost is able to deliver cutting-edge performance across a range of domains. This versatile machine learning framework boasts a crucial advantage in its ability to effectively manage categorical variables with high cardinality and handle missing data, making it a valuable tool for real-world datasets  are becoming increasingly popular in HSI due to their remarkable ability to accurately predict the concentrations of chemical components in a sample . EssentiThe 1DCNN architecture offers a notable advantage in handling large amounts of spectral data, making it well-suited for HSI applications with numerous spectral bands per pixel. This capability enables efficient processing of extensive datasets, resulting in highly accurate predictions. In this study, we employed a 1DCNN framework to predict the concentrations of puerarin, puerarin apioside, daidzin, daidzein, and starch in PTR, as shown in 2.62) and residual predictive deviation (RPD).This study employed several evaluation metrics to assess the accuracy and reliability of the models, including root mean squared error (RMSE), mean absolute error (MAE), coefficient of determination  5.3 software from ITT Visual Information Solutions, Inc. in Boulder, CO, USA. The experiments for the proposed DL models were conducted on a server equipped with an Intel(R) Xeon(R) Platinum 8368 CPU @ 2.40GHz (251G RAM) and a GA100 graphics card (A100 PCIe 80GB GPU), running the Ubuntu Linux 21.04 operating system. The model’s compilation was created in the Python programming language (Python 3.7.10) and implemented using TensorFlow 2.2.0 and CUDA 11.7. During the network training, the cross-entropy loss function and the Adam optimization algorithm were utilized, while the learning rate, and batch size were set to 0.001 and 16, respectively. The dataset was randomly split into a training set (80% of the dataset) and a test set (20% of the dataset). To ensure the reliability of the results, all experiments were conducted and averaged over 10 independent runs.33.1The contents of isoflavones and starch in PTR samples were determined and presented in 3.22 groups, which are associated with the presence of proteins and amino acids in PTR . On the other hand, CatBoost exhibited the best prediction accuracy for puerarin, puerarin apioside, and daidzin contents, with the lowest values of RMSE  and MAE , as well as the highest RPD  and R2 .Furthermore, Overall, the combination of HSI and traditional machine learning techniques has proven to be a valuable approach for predicting the levels of isoflavones and starch in PTR. Among the findings of the experiment, CatBoost demonstrated superior performance compared to the other investigated models. Nevertheless, there is still room for further improvement in the predictive accuracy of these models. One promising direction for enhancement is the incorporation of advanced wavelength selection methods.3.3.22 values above 0.90 and RPD values exceeding 3.20 for the five different components analyzed in PTR.In subsection 3.3.1, it has been demonstrated that the SG yields the best preprocessing results. Therefore, in following experiment, only the SG method was employed as the preprocessing technique. The outcomes of predicting the isoflavones and starch contents based on full wavelengths using the 1DCNN model are presented in 2 and RPD, respectively. Similarly, for puerarin apioside, the 1DCNN model surpassed the other models, yielding an average increase of 0.0696 and 0.6653 in R2 and RPD, respectively. Furthermore, the 1DCNN model exhibited enhanced prediction results for daidzin and daidzein, resulting in average increases of 0.0566, 0.0673 and 0.6226, 0.6252 in R2 and RPD, respectively. Likewise, the 1DCNN model showcased superior performance in predicting starch, with average increases of 0.0359 and 0.4598 in R2 and RPD, respectively.In terms of predicting puerarin, the 1DCNN model demonstrated improved performance compared to the traditional models, with an average increase of 0.0546 and 0.7804 in RThe regression results for the reference and predicted values of the four isoflavones and starch contents are shown in 3.4In this section, three methods, namely SPA, CARS, and UVE, were employed to select effective wavelengths, aiming to improve the prediction performance of the models. For the prediction of puerarin, the three methods selected 14, 19, and 25 important variables from the full range of wavelengths. Similarly, for the prediction of puerarin apioside, 10, 17, and 22 significant wavelengths were chosen. For daidzin and daidzein, SPA, CARS, and UVE selected 16, 31, and 33, as well as 12, 23, and 28 important variables, respectively, from a pool of 288 variables. Lastly, 13, 28, and 30 significant wavelengths were chosen for starch content prediction.The specific distribution of the selected feature wavelengths for each component prediction is shown in 2, RPD, RMSE, and MAE were determined to be 0.8703, 2.783, 0.0775, and 0.0640, respectively. Conversely, for starch prediction, SG-SPA-PLSR exhibited the highest prediction accuracy, with the lowest RMSE (10.667) and MAE (7.533), as well as the highest RPD (2.9692) and R2 (0.8876).In addition, 3.5The constituents and their corresponding concentrations are quintessential to determine the intrinsic quality and commercial value of a material. In this study, we employed a combination of HSI and data analysis techniques to accurately predict the content of key compounds in PTR. Through a comprehensive analysis of the results, it was evident that the integration of HSI with a deep learning model achieved satisfactory predictive performance. Given its convenience, non-destructiveness, and high-efficiency, this approach could serve as a viable and beneficial alternative to complicated physical and chemical procedures. It could be implemented in off‐line rapid analysis or on‐line quality control for quality evaluation of PTR.First, among the traditional algorithms compared in this study, the performance of PLSR algorithm was found to be relatively stable and moderate overall, which is consistent with its frequent utilization in similar studies involving hyperspectral prediction . However2 for component prediction reaches 0.9, even without the need for wavelength selection. This remarkable performance can be attributed to the autonomous learning capability of deep learning models, which effectively capture intricate mapping relationships between spectra and components, unaffected by spectral redundancy and noise. These findings are consistent with previous research studies  with a 1DCNN model. Our results unequivocally demonstrated that the combination of HSI and 1DCNN yielded superior predictive performance, with an average ROverall, our findings highlight the tremendous potential of HSI coupled with a deep learning model in simultaneously determining multiple key components in PTR. Considering the advantages of spectral detection, this approach offers a viable and efficient solution for non-destructive quality control assessment of PTR, which can be applicable to both off-line and on-line settings. Furthermore, future research can delve into advanced deep learning techniques like attention mechanisms to boost predictive capabilities and broaden HSI applications in quality assessment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.HH: Data curation, Writing – original draft. TW: Investigation, Writing – review & editing. YW: Data curation, Writing – review & editing. ZX: Methodology, Writing – review & editing. SC: Data curation, Writing – review & editing. LF: Data curation, Formal Analysis, Writing – review & editing. HX: Writing – review & editing. XM: Writing – review & editing. LH: Funding acquisition, Investigation, Writing – review & editing.


Horses are high-level athletic athletes prone to musculoskeletal injuries. Tendon/ligament injuries are the most frequent types of injuries which that are very difficult to treat. Instead of tissue regeneration, usually, fibrous scar tissue develops which leads to decreased functionality of the injured area and threatens the participation of sport horses. The aim of regenerative medicine is to find a treatment that promotes tissue regeneration and that allows the equine patient to return to the same level of athletic performance in the shortest time period possible. In this study, we developed a solution of equine synovial membrane stem cells and autologous serum, to be injected at the lesion site to promote tissue regeneration. We describe the processes of tissue collection, preparation, isolation of synovial stem cells, expansion, culture, cryopreservation, and posterior preparation with autologous serum. The solution was tested in 16 tendons and ligaments of equines. After treatment, all equine patients underwent a physical rehabilitation program and were monitored with physical and ultrasonographic exams. The results were very promising, and thus, support the use of equine synovial stem cells and autologous serum in the treatment of tendonitis and desmitis.Tendon and ligament injuries are frequent in sport horses and humans, and such injuries represent a significant therapeutic challenge. Tissue regeneration and function recovery are the paramount goals of tendon and ligament lesion management. Nowadays, several regenerative treatments are being developed, based on the use of stem cell and stem cell-based therapies. In the present study, the preparation of equine synovial membrane mesenchymal stem cells (eSM-MSCs) is described for clinical use, collection, transport, isolation, differentiation, characterization, and application. These cells are fibroblast-like and grow in clusters. They retain osteogenic, chondrogenic, and adipogenic differentiation potential. We present 16 clinical cases of tendonitis and desmitis, treated with allogenic eSM-MSCs and autologous serum, and we also include their evaluation, treatment, and follow-up. The concerns associated with the use of autologous serum as a vehicle are related to a reduced immunogenic response after the administration of this therapeutic combination, as well as the pro-regenerative effects from the growth factors and immunoglobulins that are part of its constitution. Most of the cases (14/16) healed in 30 days and presented good outcomes. Treatment of tendon and ligament lesions with a mixture of eSM-MSCs and autologous serum appears to be a promising clinical option for this category of lesions in equine patients. Tendonitis and desmitis are defying clinical challenges in equine patients that require long recovery periods, and ineffective tendon repair can affect their sport careers. Tendons operate near their functional limits during maximal exercise, and their ability to adapt to stress and self-repair is limited. A controlled exercise program alone or in combination with a variety of conservative treatments, such as corrective shoeing and nonsteroidal anti-inflammatory drugs (NSAIDs), is still the gold standard therapy for equine tendon disease . CurrentThe aim of tendinopathy treatment is to achieve tissue regeneration and return to complete organ function and performance. Recently, tissue engineering approaches have attracted attention for tissue repair. Among the approaches, the use of mesenchymal stem cell-based therapy has increased, since it is a promising approach for tissue repair and regeneration including tendinopathy and desmitis ,3,4,5,6.Mesenchymal stem cells (MSCs) can be isolated from several tissue sources such as bone marrow, peripheral blood, dental pulp, umbilical cord, and amniotic fluid . MSC chaSynovial membrane mesenchymal stem cells (SM-MSCs) were initially isolated, in 2001, by De Bari et al. , from huSM-MSCs have higher chondrogenic capacity than other studied sources of MSCs, such as bone marrow (BM-MSCs) ,13. CartIn a recent study, using a rabbit model, Bami et al. highlighted the superiority of SM-MSCs in terms of chondrogenesis, osteogenesis, myogenesis, and tenogenesis . A studySM-MSCs have been defined as MSCs due to their phenotypic profile and differentiation potential. Even though there are no specific antibody markers to identify these MSCs, there is general agreement that MSCs should be negative to hematopoietic markers CD34 and CD45 and positive to CD44, CD73, CD90, and CD105 . MochizuIn 2003, Fickert et al. reported that the markers CD9, CD44, CD54, CD90, and CD166 could be used to identify MSCs isolated from the synovium of human patients with osteoarthritis (OA), and they also confirmed that CD9/CD90/CD166 triple-positive cell subgroups had obvious chondrogenic and osteogenic differentiation abilities .Prado et al. confirmed the mesenchymal nature of equine synovial membrane and fluid-derived stem cells through the expression of significant hematopoietic , mesenchymal , pluripotency (OCT3/4 and NANOG), embryonic (Tra-1-81), inflammatory, and angiogenesis  and LY6a) markers . AlthougNevertheless, the immunophenotype characterization of equine MSCs (eMSCs), as well as in other veterinary species, has not yet been completely established . This isHorses are high performance athletes prone to musculoskeletal diseases, i.e., osteoarticular, as well as tendon/ligament lesions and fractures of various degrees due to sport- and age-related injuries. These pathologies resemble human musculoskeletal conditions, and therefore, horses are a valuable animal model for assessing stem cell and cell-based therapies prior to the translation of results into humans . The useRecent studies have suggested that MSCs can self-renew, migrate to injury sites (homing), perform multilineage differentiation, and secrete bioactive factors, thus, increasing proliferation and migration of tendon stem/progenitor cells via paracrine signaling and increasing the regeneration ability of tissues with poor aptitude ,5,22,23.In fact, the knowledge of the importance of this paracrine action has opened doors to cell-free therapeutic strategies in regenerative medicine. The soluble factors  and nonsoluble factors (extracellular vesicles and exosomes) released in the extracellular space by MSCs, commonly known as secretome, have become the focus of novel therapeutic approaches due to their key role in cell-to-cell communication, their active influence on immune modulation, and their pro-regenerative capacity both in vitro and in vivo . TherefoIn the present study, equines used as show jumping and dressage athletes as well as leisure horses with acute and chronic lesions were treated with intralesional administration of the considered combination, i.e., autologous serum and eSM-MSCs. The treatment consisted of two injections, 15 days apart. Pre- and post-treatment evaluations consisted of clinical, orthopedic, and tendon/ligament ultrasound exams. None of the selected equine patients had previously received any other regenerative treatment.This prospective longitudinal study was performed in Portugal between February 2016 and January 2019. Sixteen horses, from 5 to 22 years old with acute and chronic signs of lameness were enrolled in this study , whose sport activities were distributed over show jumping (14), dressage (1), and leisure (1). The horses were all outpatients from an equine ambulatory clinic. This study included the treatment of 16 tendons, i.e., 14 superficial digital flexor tendons and 2 deep digital flexor tendons, and 4 suspensory ligaments. Lameness was scored based on the American Association of Equine Practitioners (AAEP) scale  and confIn this study, horses with acute or chronic lameness , with diThis study was carried out in accordance with the Organismo Responsável pelo Bem Estar Animal (ORBEA) from ICBAS-UP, project number P289/ORBEA/2018 recommendations and authorization. Treatments were performed with permission and signature of an informed consent from the equine patient’s legal trainer, following a thorough explanation on the procedure itself and possible risks and associated effects, in accordance with national regulations and project approval from the competent authorities. In addition, no animals were euthanized for this study.6 cells/mL and 1 mL of autologous serum in a total volume of 2 mL).The eSM-MSC donor was a young and healthy foal, 7 months old, who died accidentally when running in the arena. The trainer authorized synovial membrane collection from the hocks, knees, and fetlocks. The synovial membrane was evaluated and its appearance was transparent, bright, and smooth; in addition, the presence of viscous and transparent synovial fluid confirmed its soundness. The skin covering the incisional field was surgically cleaned with chlorohexidine and alcohol. The skin and subcutaneous tissue were incised, debrided, the articular capsule was opened, and the synovial membrane was isolated and extracted into a Dulbecco′s phosphate buffered saline (DPBS) container. The samples were transported to the laboratory with ice packs for refrigerated temperatures. ® . Fresh tissue was transported to the laboratory facilities in a hermetically sealed sterile container in transport media (supplemented with 3% (v/v) penicillin-streptomycin  and 3% amphotericin B (Gibco®) and processed within a period of up to 48 h. The synovial tissue was digested using collagenase and the isolated cells were incubated in a static monolayer culture using standard MSC basal medium supplemented with 10% fetal bovine serum (FBS) and maintained in standard culture conditions  until they reached confluence. Cells from confluent cultures were cryopreserved in 10% dimethylsulphoxide (DMSO) and FBS, at a concentration of 3 × 10⁶ cells/mL, using a control rate temperature freezer . For expansion optimization, cells were cryopreserved in passages (P) between P2 and P3 to generate suitable master cell banks (MCBs). Expansion, thereafter, was analyzed during a maximum of 20 cumulative population doublings (cCPDs). The range of cCPDs chosen allowed for enough expansion to maximize the number of cells in the working cell banks (WCB) but kept the cCPDs within the genomic stability range.After collection, the equine synovial membrane was prepared at the Laboratory of Veterinary Cell-based Therapies from ICBAS-UP. The isolation protocol for eSM-MSCs was developed by patented proprietary technology RegeneraFor all the differentiation protocols, cells in P4 were used after thawing.4 cells/cm2 were seeded in the wells of a 12-well plate , with the addition of the standard culture medium. The plate was incubated under standard conditions for 4 days. After this period, the culture medium of 10 wells was replaced by complete adipogenesis differentiation medium , 2 wells were used as controls and maintained with the standard culture medium. Following the manufacturer’s instructions, the media were replaced every 3–4 days and the cells maintained in differentiation for 14 days. At the end of this period, the oil red O staining protocol was performed using a handmade solution. The culture differentiation medium was removed, and the wells were gently washed with PBS. Cells were fixed with 4% formaldehyde  for 10 min at room temperature, and the wells were washed 3 additional times with phosphate-buffered saline (PBS). Oil red O solution was added to each well and the plate incubated for 10–20 min at room temperature. Oil red O was discarded, and any excess dye was removed by several washes with PBS. PBS was added to each well for visualization. The aim of this assay was the identification of rounded cells with intracytoplasmic lipid vacuoles and their red coloration due to the exposure to the oil red O solution.For the adipogenic differentiation protocol, 1 × 107 viable cells/mL. To generate micro-mass cultures, 5 μL droplets of the cell suspension were placed in the center of 10 wells of a 96-well plate  to induce chondrogenic differentiation. The plate was maintained under standard conditions for 2 h. After this time, chondrogenic differentiation medium  was added to 8 wells; the other 2 wells were considered to be controls and to these, the standard culture medium was added. Following the manufacturer’s instructions, the media were replaced every 3–4 days and cells maintained in differentiation for 14 days. At the end of this period, the Alcian blue staining, pH 2.5, protocol was performed . The culture differentiation medium was removed, and the wells were gently washed with PBS. The cells were fixed with 4% formaldehyde for 20 min at room temperature, and the wells were washed 3 additional times with PBS. Alcian blue solution was added to each well and the plate incubated for 30 min at room temperature. Then, the Alcian blue was discarded, and the wells were rinsed 3 times with 3% acetic acid (v/v). For neutralization of acidity and for visualization by inverted phase contrast microscopy, distilled water was added to all wells. The aim of this assay was the identification of chondrogenic aggregates and their coloration in blue due to the exposure to Alcian blue solution.Thawed eSM-MSCs were automatically counted, and cell viability determined (%). Then, the cells were centrifuged, supernatant removed, and the pellet resuspended in culture medium to generate a cell suspension with 1.6 × 103 cells/cm2 were seeded into the wells of a 12-well plate. The plate was maintained under standard conditions for 4 days. After this period, the culture medium of 10 wells was replaced by complete osteogenic differentiation medium , and 2 wells were used as controls and maintained with the standard culture medium. Following the manufacturer’s instructions, the media were replaced every 3–4 days and the cells maintained in differentiation for 21 days. At the end of this period, the alizarin red s staining protocol was performed using a commercial solution . The culture differentiation medium was removed, and the wells were gently washed with PBS. The cells were fixed with 4% formaldehyde for 30 min at room temperature, and the wells were washed twice with distilled water. One ml of 40 mM of alizarin red solution was added to each well and the plate incubated for 30 min. Then, the alizarin red solution was discarded, and the wells were rinsed 3 times with distilled water until the supernatant became clear. For visualization by inverted phase contrast microscopy, PBS was added to all the wells. The aim of this assay was to identify calcium-containing osteocytes stained in red after exposure to alizarin red solution. For osteogenic differentiation, 8 × 10® Colcemid™ Solution, Gibco®, Waltham, MA, USA). After 4 h, the eSM-MSCs were collected and resuspended in 8 mL of 0.075 M KCl solution, followed by incubation under standard conditions for 15 min. After centrifugation (1700 rpm), 8 mL of ice-cold fixative comprising methanol and glacial acetic at a proportion of 3:1 was added and mixed. Afterwards, the cells were centrifuged again. Three fixation rounds were carried out. After the last centrifugation, the suspension of eSM-MSCs was spread over glass slides. A karyotype analysis was performed by one scorer on Giemsa-stained cells. For the different passages, a specific number of cells in metaphase were evaluated depending on the number of cells with a normal karyotype identified, guaranteeing a better representation of the population under study.The eSM-MSCs in two different passages (P4 and P7) were submitted to cytogenetic analysis to determine the genetic stability in terms of chromosome number and occurrence of neoplastic changes. For both passages, 70–80% confluence was reached. Then, the culture medium was changed and supplemented with 10 μg/mL colcemid solution  culture medium was added to the culture flasks, which were then incubated under standard conditions. The culture medium rich in factors secreted by the cells (CM) was collected after 48 h. The collected CM was then concentrated five times. After collection, it was centrifuged for 10 min at 1600 rpm, and its supernatant collected and filtered with a 0.2 μm syringe filter . For the concentration procedure, Pierce™ Protein Concentrator, 3k MWCO, 5–20 mL tubes  were used. Initially, the concentrators were sterilized following the manufacturer’s instructions. Briefly, the upper compartment of each concentrator tube was filled with 70% ethanol (v/v) and centrifuged at 300× g for 10 min. At the end of the centrifugation, the ethanol was discarded, and the same procedure was carried out with DPBS. Each concentrator tube was subjected to two such centrifugation cycles, followed by a 10 min period in the laminar flow hood to complete drying. Finally, the upper compartment of the concentrator tubes was filled with plain CM (1 × concentration) and subjected to new centrifugation cycles, under the conditions described above, for the number of cycles necessary to obtain the desired CM concentration (5×). The concentrated CM was stored at −20 °C and subsequently subjected to a Multiplexing LASER Bead analysis  to identify a set of biomarkers present in the Equine Cytokine 8-Plex Assay (EQCYT-08-501). The list of searched biomarkers includes basic fibroblast growth factor (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), interleukins (IL) IL-6, IL-8, IL-17A, and human growth-regulated oncogene/keratinocyte chemoattractant (GRO/KC). All samples were analyzed in duplicate.The eSM-MSCs were harvested from equine synovial membrane and maintained in culture, as previously described. The cells in P4 were subjected to an analysis of their conditioned medium (CM) to identify cytokines and chemokines secreted after conditioning. When in culture, after reaching a confluence of around 70–80%, the culture medium was removed, and the culture flasks were gently washed with DPBS two to three times. Then, the culture flasks were further washed two to three times with the basal culture medium of each cell type, without any supplementation. To begin the conditioning, non-supplemented DMEM/F12 GlutaMAX™ . Consecutive sections were cut at 2 μm, deparaffinized, hydrated, and submitted to immunohistochemical analysis using the Novolink™ Polymer Detection Systems  kit, according to the manufacturer’s instructions. Information regarding the primary antibodies and antigen retrieval recovery methods used in this study is summarized in Early passages of eSM-MSCs-P0 and -P3 were maintained in culture until a confluence of 70–80% was reached, and then enzymatic detachment was performed with 0.25% trypsin-EDTA solution. A cytoblock was performed fixing the cells with Sure ThinThe antibodies were selected to confirm the pluripotent and mesenchymal origin of the eSM-MSCs’ octamer-binding transcription factor 4 (OCT4), homeobox protein (NANOG), proto-oncogene receptor tyrosine kinase or stem cell factor receptor (c-kit), synovial origin (lysozyme), and non-epithelial origin histogenesis (vimentin). Additionally, pan-cytokeratin (AE1 and AE3), synaptophysin, CD31, and glial fibrillary acidic protein (GFAP) were used to confirm there were no vascular, epithelial, neuronal, and neuroendocrine origins of cells, respectively. For each antibody, appropriate negative and positive controls were included, and all primary antibodies were incubated overnight.®, Tokyo, Japan) and the software Imaging Software NIS-Elements F Ver4.30.01 . A semi-quantitative score was used for mmunoexpression evaluation, consisting of the percentage of labeled cells  and labeling intensity . Immunoreactivity was considered positive when distinct nuclear and cytoplasmic staining was recognized in at least 5% of the cells. The final step consisted of microscopic cell observation, evaluation, and photograph using the microscope Eclipse E600  using an automatic counter . Then, the cell number was adjusted to 5 × 10⁶ cells/mL, and then 2 mL of the solution of eSM-MSCs suspended in autologous serum was transferred to a perforable capped vial and preserved on ice until the time of administration.The eSM-MSC solution for local clinical application in the 16 equine patients, was a combination of allogenic eSM-MSCs suspended in autologous serum. Prior to preparation of the final therapeutic combination, autologous serum was isolated from whole blood. Then, 10 mL samples of whole blood were collected into dry blood collection tubes, and after clotting, they were centrifuged at 2300 rpm for 10 min and their supernatant (serum) collected and transferred to a 15 mL falcon. Then, the serum was inactivated through a water bath at 56 °C for 20 min followed by cooling on ice. Finally, the serum was centrifuged and filtered using a 0.22 µm syringe filter and stored at −20 °C until further use. Cryopreserved P3 eSM-MSC batches were thawed in a 37 °C water bath, and the content was transferred to a 10 mL tube with autologous serum and slowly diluted, followed by the addition of sterile DPBS until reaching 10 mL. Then, the mixture was centrifuged at 1600 rpm for 10 min. The supernatant was discarded, and the cell pellet was re-suspended in a mixture of autologous serum at a ratio of 0.8:1. Cell counting and viability were determined by using the trypan blue exclusion dye assay , orthopedic examination , and flexion test of the main joints for 60 s followed by trot). Lameness was evaluated at a walk and a trot on hard surface and scored on a scale from 0 to 5, according to the AAEP parameters. Complementary diagnostic exams included regional nerve blocks (to identify the pain area), radiographs, and ultrasound image as reported in other studies ,30,31,32Following the assumptions of the exclusion criteria, the horses did not receive any treatment before or after the administration of the therapy protocol. In the case of adverse events occurring, such as inflammatory/anaphylactic reactions or infections, the horses should be immediately evaluated and treated with anti-inflammatories or antibiotics, in accordance with their clinical status. The equine patients were monitored in the 48 h after treatment and any occurrences were registered. Following the treatment, the equine patients were assessed periodically to control the equine patient’s healing evolution and to provide valid comparative data among equine patients within the same study group. Selected horses were sedated with detomidine (0.02 mg/kg), trichotomized, a regional nerve block was performed with lidocaine 2% , and the surgical skin was disinfected with chlorohexidine and alcohol. The therapeutic combination was aspired to a 2 mL syringe and homogenized, ultrasound was used to identify the lesion site, and an ultrasound guided injection was performed at the lesion over three different points. Finally, a bandage was applied to the limb. All equine patients were injected with phenylbutazone  at the end of the treatment. The established protocol included a second eSM-MSC administration 15 days after the first treatment using the same protocol.®, Shenzhen New Industries Biomedical Engineering Co Ltd., Shenzhen, China). For each assessment, a complete examination of the structure was conducted by means of longitudinal and transverse scans. The obtained images were evaluated at each examination for two parameters: lesion echogenicity and lesion longitudinal fiber alignment (FA). The contralateral healthy limb was used as comparison. The evaluation was performed on the treatment day (Day 1) as well as on Days 15, 30, and 45 post-treatments, as presented in Tissue regeneration was estimated through a lameness evaluation, pain to pressure test, limb inflammation, sensitivity, and ultrasound image . Lesion ultrasonographic evaluations were performed using a 7.5 MHz linear transductor probe , as shown at The analysis of CM revealed the production and secretion of several factors with immunomodulatory functions, capable of intervening beneficially in tissue regeneration. The results of the eSM-MSC CM analysis are shown in The eSM-MSCs showed strong expressions of OCT4/NANOG, vimentin, and lysozyme which confirmed marked stem cells, non-epithelial cells, and synovial cells, respectively; weak expression of GFAP; and no expression of CD31, synaptophysin, and pan-cytokeratin, as seen in No horse had any adverse event that required study cessation, unplanned procedures, or additional treatments. All intra-tendinous injections and follow-up procedures had no adverse reactions , as shown by Godwin et al. (2012) . No horsTendon/ligament regeneration occurred in a time frame of less than 30 days in 80% of the cases and between 30–90 days in 20% of the cases. In this study, eight horses had a lesion on the right front limb, six horses had a lesion on the left front limb, and two horses had a lesion on the right hind limb. There were 14 acute cases and two chronic cases. Chronic cases were diagnosed 6 months before our approach.After Day 90, meaning they had completed the proposed rehabilitation physical program, the horses started cantering and started to return to their usual work plan. By Day 120 post the first treatment, 87.5% of the horses were back to full work, with the exception of the 12.5% who needed another 30 days to return to full work. All horses returned to the same level of sport activity they had before injury. Radiograph exams were performed to rule out the presence of other associated pathologies and regional nerve blocks were performed to better localize the injured region originating the pain.Ultrasound images at Day 1 and at Day 30 clearly illustrate the evolution of tendon regeneration. Changes in echogenicity, fiber alignment, and cross-sectional area are evident, as seen in Recently, eSM-MSCs have become an interesting subject for those who study cellular and cell-based therapies due to their promising ability to promote tissue regeneration with high capacity of regeneration of articular structures, tendons, and ligaments. Regarding the collection, isolation, expansion, freezing, and thawing protocols used in this clinical trial, it was possible to use these cells in equine tendon regenerative treatments. The full characterization of eSM-MSCs presents a significant challenge since eSM-MSCs are not as well studied as MSCs from other species, namely human MSCs. However, in this study, their stemness and origins were confirmed through different processes: trilineage differentiation, karyotype, secretome, and immunohistochemistry. All the SM-MSC cultures presented monolayer culture, plastic adherence capacity, and fibroblast-like shape ,41,42,43The karyotype presented some genomic variations when the number of passages was increased. That was consistent with some studies regarding genomic variations along cell passages ,46,47,48As reported by Simona Neri, the interpretation of genetic instability and senescence of cultured MSCs is controversial, but the increasing incidence of genetic alterations at advanced culture times clearly indicates that few culture passages correspond to a reduced chance to harbor dangerous alterations. Therefore, prudent behavior is desirable with a reduction in culture times as much as possible to avoid safety concerns . More stDuring the last decade, it has been shown that the therapeutic effectiveness of MSCs is due mainly to the release of paracrine factors, namely CM, composed of soluble  and nonsoluble factors (extracellular vesicles) that are primarily secreted in the extracellular space by stem cells . CM’s paIn our study, eSM-MSCs revealed a CM with a high level of KC/GRO, MCP-1, Il-6, FGF-2, G-CSF, GM-CSF, and IL-8. This highlights the intense activity of fibroblasts, producing KC/GRO that is chemotaxic for neutrophils during inflammation. MCP-1 is essential for reperfusion and the successful completion of musculoskeletal tissue after an ischemic injury . MacrophA recent study highlighted that hematopoietic factor promoted tendon healing in aged mouse tendons. Histochemical results demonstrated that vascularization of the injury site was significantly elevated. It was concluded that vascular endothelial growth factor (VEGF) played an important role in decreasing adipocyte accumulation and also improved vascularization of the tendon during aged tendon healing. Active regulation of VEGF may improve the treatment of age-related tendon diseases and tendon injuries .Studies with human BM-MSCs using a human-specific proteome profiler array with different angiogenic factors such as VEGF-A, IL-6, IL-8, platelet-derived growth factor A (PDGF-A), endothelin-1 (ET1), and urokinase plasminogen activator (uPA), which had not been previously reported in the CM of human MSCs, were also identified in an equine array, confirming what we found in this study . This faA study using a murine osteoarthritis (OA) model demonstrated that an injection of MSCs CM, similarly to injection of MSCs, resulted in early pain reduction and had a protective effect on the development of cartilage damage in a murine OA model, by using the regenerative capacities of the MSCs-secreted factors .Interestingly, the results accumulated so far have provided evidence that veterinary patients affected by naturally occurring diseases should provide more reliable outcomes of cell therapy than laboratory animals, thus, allowing translating potential therapies to the human field. More recently, a cell-free therapy based on MSCs CM has been proposed. Even though there are very few clinical reports to refer to in veterinary medicine, recent acquisitions suggest that MSC-derived products may have major advantages compared to the related cells, for example, they are considered safer and less immunogenic . As evidIn this study we used a cell-based therapy instead of CM itself, but we were aware of its effect and potential on cell-based therapies; its advantages and therapeutic effects were the reason why this study was performed.To better characterize the cells under study, we performed immunohistochemistry assays. The choice of markers was based on a previous work  and inclThe immunohistochemistry analysis showed the absence of CD31, sinaptophysine, and pan-cytokeratin expressions, confirming no vascular, neuronal and epithelial origins of cells. GFAP was weakly expressed, being less expressed in P3 than in P0 cells. CD31 was performed to investigate the presence of hematopoietic cells in eSM-MSCs. The expression of VEGF was not found, these results being similar to those from Fulber et al., and to other authors that evidenced the absence of hematopoietic markers ,85. The In our clinical trial, we treated mainly early acute lesions; 87.5% of the cases were acute lesions of tendons or ligaments. Therefore, we created a master cell bank of allogenic eSM-MSCs suitable for treatments in early acute phases versus treatments with autologous cells where time of tissue collection, preparation, and cell culture need to be considered. Furthermore, cell harvesting for autologous treatment is an invasive procedure which is unnecessary with this new eSM-MSC solution. The possibility of having a master cell bank enables faster healing of the organ and a quicker return to sport life. Horses spend less time in recovery time and have a regenerated tissue instead of a fibrotic tissue. These are some advantages of the eSM-MSC solution. Another concern is that in the early stages of the lesion there is an inflammatory phase; however, the paracrine factors released by eSM-MSCs also have anti-inflammatory action, reducing inflammation. Chronic cases represented 12.5% of the cases, involving four structures. Three of the horses recovered in 30 days and one of the horses had a delayed recovery time.The delayed recovery time in 20% of the structures, meaning 12.5% of the horses, was due to, in Case 6, an increased number of involved structures (more than one tendon or ligament) and a foot conformation abnormality, as the horse had a fetlock hyperextension that was impairing correct tendon healing. This was corrected with special shoeing. Inappropriate rehabilitation program (Case 7) was another cause of delayed recovery time. As soon as the corrective shoeing was performed, ligament regeneration started.We could also conclude that lameness grade was not directly correlated with lesion cross-sectional area. Horses with ultrasonographic cross-sectional grade 1, 2, and 3 lesions presented lameness grade 4/5, which was observed in 9 of 16 patients. Lameness grade 3/5 was presents in 4 of 16 of equine patients with ultrasonographic cross-sectional grade 1 and 2 lesions. Lameness grade 2/5 was present in 3 of 16 equine patients with ultrasonographic cross-sectional grade 1 lesions.Kamm et al. (2021) concluded that based on the evidence to date, tendons appear to have improved healing when treated with allogeneic MSCs, and the use of these treatments in equine tendon and ligament lesions is warranted . ColbathTo sum up, this study accomplishes the criteria for reporting veterinary and animal medicine research for MSCs in orthopedic applications  and the From a clinical point of view, the idea of having an allogenic eSM-MSC cell bank is very interesting. Therefore, the possibility of having a universal donor who can provide a large amount of eSM-MSCs, to culture and preserve non-immunogenic cells whose availability is immediate, allowing a quick and effective therapeutic answer in acute stages of musculoskeletal lesion is the paramount goal of orthopedic medicine.From a “one-health” perspective, equines play an important role as a model for human musculoskeletal disorders; the high-level analogy between human and equine structures may have a great translational value for both species for future clinical aspects ,90. TherBased on the clinical, ultrasonographic, and performance outcomes identified in the present study, the use of eSM-MSCs together with autologous serum solution has proven its efficiency for tendon and ligament repair and contributes to reduce the recovery period and subsequent rapid return to athletic activity. The therapy was demonstrated to be safe and had no adverse findings. The clinical results and athletic outcomes of the horses were very positive. Comparing our study with others, using for example BM-MSCs, it seems that our new approach has shorter recovery times and fewer re-injuries ,77. Thes


During the follow-up, 18 patients had a CV event, and 37 patients had a renal event. In the univariate cox regression analysis, systolic AOBP was found to be predictive of the primary outcome , and after adjustment for eGFR, smoking status, diabetes, and a history of CV disease and systolic and diastolic AOBP were also found to be predictive of the primary outcome . Conclusions: In patients with CKD, AOBP appears to be prognostic of CV risk or risk for kidney disease progression and could, therefore, be considered a reliable means for recording BP in the office setting.Background: The aim of this study was to evaluate the prognostic value of automated office blood pressure (AOBP) measurement in patients with hypertension and chronic kidney disease (CKD) stage 3–5 not on dialysis. Methods: At baseline, 140 patients were recruited, and blood pressure (BP) measurements with 3 different methods, namely, office blood pressure (OBP), AOBP, and ambulatory blood pressure measurement (ABPM), were recorded. All patients were prospectively followed for a median period of 3.4 years. The primary outcome of this study was a composite outcome of cardiovascular (CV) events  or a doubling of serum creatine or progression to end-stage kidney disease (ESKD), whichever occurred first. Results: At baseline, the median age of patients was 65.2 years; 36.4% had diabetes; 21.4% had a history of CV disease; the mean of estimated glomerular filtration rate (eGFR) was 33 mL/min/1.73 m The incidence of end-stage kidney disease (ESKD) keeps rising in many industrialized nations, regardless of the widespread use of treatments to limit the progression of CKD . It has se study . Hypertese study . HoweverSimilarly to the general population, the diagnosis of hypertension in patients with CKD is based on office blood pressure measurements (OBP) and, in recent decades, ambulatory blood pressure measurements (ABPM). The latter method was found to be more accurate, not only in the identification of hypertension but also in the prediction of CVD risk , despiteAOBP correlates better than OBP with left ventricular mass  and micrThe participants of this study were recruited at the nephrology clinics of two Greek hospitals, Athens General Hospital Evangelismos and University General Hospital of Alexandroupoli. The inclusion criteria were patients with hypertension who were under treatment, had CKD stage 3–5 (according to the CKD-EPI equation), were not on dialysis, and were between 18 and 85 years of age. Exclusion criteria included acute kidney injury within the previous 2 months; kidney transplantation; pregnancy; hemodialysis or peritoneal dialysis; a change in antihypertensive therapy during the previous 15 days; and serious non-cardiovascular diseases such as cancer or liver cirrhosis. Those unable to attend follow-up office visits were omitted as well.All eligible individuals received a medical interview, anthropometric measurements, and blood and urine tests at the start of the study. Specifically, laboratory data  were collected during the initial study visit. Each participant’s history of smoking, hyperlipidemia, and risk factors for CVD had been assessed. Serum creatinine concentrations were measured, and the estimated glomerular filtration rate (eGFR) was obtained by using the CKD-EPI equation adjusted for body surface area. This study was conducted in conformity with the principles of the Declaration of Helsinki. All participants gave their informed consent, and the study was authorized by the hospitals’ ethical committees and scientific boards.All BP readings  were taken in hospital outpatient settings. OBP was computed as the mean of two consecutive measurements by using a certified digital oscillometric BP electronic instrument  and a medium–large stable bladder, 22–42 cm. BP measurements were obtained at 1 min intervals from 8:30 to 10:30. Before assessing OBP, participants were instructed to stay seated for 5 min and to avoid speaking; a doctor from the research team was present throughout. Systolic blood pressure (SBP) lower than 140 mm Hg and diastolic blood pressure (DBP) lower than mm Hg were deemed acceptable levels for OBP . After tFor 24 h ABPM monitoring, a certified electronic Microlife WatchBP O3, bladder size 22–32 cm, or 32–42 cm, was employed. The device was set to take a reading every 15 min during the day (06:00–23:00) and every 30 min during the night (00:00–06:00) (23:00–06:00). At the time of measurement, participants were told to immobilize their arm and record their day activities and sleep duration in a notebook. SBP lower than 135 mm Hg and DBP lower than 85 mm Hg were accepted values of normality for ABPM in adults during daytime (awake); night-time (asleep) SBP and DBP lower than 120 and 70 mm Hg, respectively; and 24 h SBP and DBP lower than 130 and 80 mm Hg, respectively .The Microlife WatchBP Office device, which was validated for OBP measurement by the International Protocol , was useBeginning with the initial study visit, participants attended follow-up meetings every six months to monitor BP management, antihypertensive therapy, and primary outcomes. The primary outcome of this study was a composite outcome of a CV event, serum creatine doubling, or development of ESKD, whichever came first. CV events comprise fatal or nonfatal CV events, such as myocardial infarction; coronary heart disease, including coronary intervention, hospitalization for heart failure (HF), stroke, and intermittent claudication; and peripheral arterial disease interventions, whichever happened first. Renal events were defined as serum creatinine levels doubling or the progression to ESKD. The endpoint of ESKD was met on the first day of dialysis treatment.2 test, and continuous variables were compared by using Student’s t test. We utilized Cox proportional hazard models to assess the relationship between the initial BP values  and the onset of a CV or renal event. The correlation between OBP, AOBP, and ABPM values and the primary outcome was evaluated by using hazard ratios and their 95% confidence intervals per 1 mm Hg increase in systolic and diastolic BP. In the Cox regression analysis, two models were conducted: the first model was univariate-unadjusted, and the second model was multivariate-adjusted for baseline covariates known to influence renal and CV outcomes, such as smoking status , diabetes, eGFR, and a history of CVD. Moreover, by using the Cox regression analysis, we developed survival curves, which were adjusted for smoking status , diabetes, eGFR, and a history of CVD, in various groups of patients based on normal and high levels of BP assessed at baseline by using the three distinct techniques, OBP, AOBP, and ABPM. All p values are two-sided, and p < 0.05 denotes statistical significance. All statistical analyses were performed by using version 20.015 of the MedCalc statistical tool.The Kolmogorov–Smirnov test was utilized to examine the data for normality. Normally distributed continuous variables are expressed as the mean standard deviation, non-normally distributed continuous variables are expressed as the median (interquartile range), and categorical variables are expressed as a percentage. Categorical variables were compared by using the χ(n = 9), changes in antihypertensive treatment 2 weeks before the study (n = 8), and acute kidney injury on top of CKD in the last two months (n = 4). Six patients were lost to follow-up. The median age of the patients was 68 (57–76) years, and 50 (35.7%) were women. In total, 51 patients (36.4%) had diabetes, 30 patients (21.4%) had a history of CVD, 83 patients (59.3%) were receiving drugs for hyperlipidemia, and 19 patients (13.6%) were current smokers. The number of patients with CKD stages 3a, 3b, 4, and 5 was 39 (28%), 42 (30%), 47 (33.5%), and 12 (8.5%), respectively, with a mean eGFR of 33 ± 14 mL/min/1.73 m2. Demographic, clinical, and treatment information on the 140 patients included in the cohort is presented in We included 140 of 161 eligible patients. The reasons for exclusion were inadequate ABPM recordings At baseline, the mean systolic/diastolic OBP, AOBP, and daytime ABPM were 151/81 mm Hg, 134/77 mm Hg, and 132/77 mm Hg, respectively . The meaCompared with patients without an event, patients who experienced CV or renal events during the follow-up period had lower eGFRs; higher levels of iPTH and albuminuria; higher systolic daytime, night-time and 24 h ABPM values; and higher numbers of antihypertensive drugs used .From the Cox regression analysis, neither the systolic nor the diastolic OBP measurements were predictive of the primary outcome by using the unadjusted model; however, the diastolic OBP readings were found to be predictive of the primary outcome according to the adjusted model . Progressing with the analysis, the systolic AOBP readings were predictive of CV or renal events by using the unadjusted and adjusted models . The diastolic AOBP readings were not predictive of the primary outcome when fitted to the unadjusted model but were predictive according to the adjusted model . Systolic daytime ABPM appeared to be predictive of the primary outcome by using the unadjusted model  but was not predictive with the adjusted model, and diastolic daytime ABPM was not found to be predictive by using the unadjusted model but was predictive with the adjusted model . Systolic night-time and 24 h ABPM were predictive according to the unadjusted model  and the adjusted model . On the other hand, diastolic night-time and 24 h ABPM were not predictive when fitted to the unadjusted model but were predictive according to the adjusted model . All the results are presented in p = 0.050 and p = 0.003, respectively). On the other hand, high systolic OBP and daytime and 24 h ABPM were of no prognostic importance. Regarding normal and high baseline diastolic BP, which were measured via the three methods, fitting the adjusted survival curves, it emerged that only high diastolic night-time ABPM was related to the primary outcome of this study (p = 0.004) (= 0.004) .With this prospective cohort study, we were the first to investigate the prognostic effect of AOBP measurements in hypertensive patients with stage 3 to 5 CKD who were not on dialysis. As a result, high systolic AOBP readings were found to be a significant factor for predicting the composite outcome of CV or renal events even after adjustment for smoking status, eGFR, diabetes, and a history of CVD. Furthermore, diastolic AOBP measurements were also found to be prognostic for CV or renal events when fitted to the adjusted model. Taken together, these results suggest that high systolic and diastolic AOBP readings are significant risk factors for the prognosis of hypertensive patients with CKD stage 3–5 in terms of CV and kidney disease.These findings are partially in line with those of the study of Myers et al. (2015) in a non-CKD population in which the prognostic values of AOBP readings for the development of future CV events were compared . IncludeIn a second study, which was similarly conducted in a non-CKD population (6.183 CHAP participants 66 years old on antihypertensive treatment at baseline), the association between obtained AOBP at baseline and CV events was investigated . The ave2) in the SPRINT trial, patients were randomly allocated to an SBP goal of 120 mm Hg  or 140 mm Hg  [In the SPRINT study, 9361 patients with an SBP of 130 mm Hg or more and a high CV risk were randomly given an SBP goal of less than 120 mm Hg (intensive therapy) or less than 140 mm Hg (standard therapy) [ = 1316) ; 112 indIn a comparison of OBP and AOBP, it has been demonstrated that AOBP reduces the white coat effect, prevents speaking during the rest and measurement periods, and eliminates observer error and bias ,23,24. I2) for whom BP measurements were obtained with the 3 different methods: OBP, AOBP, and ABPM [In our study comparing the mean SBP measurements with the three different methods, namely, OBP, AOBP, and ABPM, we found that the mean difference in systolic BP between AOBP and OBP was 17 mm Hg, while the difference in mean SBP between AOBP and daytime ABPM was only 2 mm Hg. These results are in concordance with the results of a cross-sectional study that included 91 CKD patients (mean eGFR 45 mL/min per 1.73 m2) [Regarding the prognostic role of the other two methods of BP measurement that were implemented in our study, systolic OBP was not predictive of the primary outcome, while diastolic OBP appeared to be prognostic of CV or renal events only when the adjusted model was applied. Systolic and diastolic ABPM were revealed to be prognostic of CV or renal events. These results are consistent with the findings of Minutolo et al. (2011), who investigated the prognostic role of daytime systolic and diastolic BP in comparison with OBP in 436 individuals with CKD  in the current study was always the same; meanwhile, a random order could be a more accurate method to compare the three techniques. In addition, home BP measurements could be employed to compare in-office and out-of-office BP measurement techniques comprehensively. Finally, our findings are based on a single BP measurement with the three different methods in two consecutive days at the beginning of the study. However, repeated measurements over time and longitudinal change could provide more a precise ability to predict outcomes.In conclusion, systolic and diastolic AOBP measurements perform well in the prediction of CV or renal risk in CKD hypertensive patients not on dialysis. The findings of this study affirm the role of AOBP as the reference technique for the assessment of BP in the office setting in patients with CKD. These results are in line with the KDIGO 2021 Clinical Practice Guideline for the Management of Blood Pressure in Chronic Kidney Disease , which p