With the increasing digitalization in health care, an effective instrument is necessary to assess health care consumers’ digital competencies—their “eHealth literacy.” The 7-scale eHealth Literacy Questionnaire (eHLQ), based on the theoretically robust eHealth Literacy Framework, has shown strong psychometric properties in Denmark and Australia.The aim of this study was to translate, culturally adapt, and evaluate the psychometric properties of the Swedish version of the eHLQ.We followed the Translation Integrity Procedure guidelines to translate and culturally adapt the questionnaire to Swedish using forward and backward translations, review by an expert panel, and cognitive interviewing. The psychometric properties of the Swedish eHLQ were investigated by evaluating its internal consistency (Cronbach α) and a priori–defined factor structure .A total of 236 primary health care patients and parents of hospitalized children were included in the validation analysis. The mean age was 48.5 years, and 129 (55%) were women. All 7 eHLQ scales showed good internal consistency, with the Cronbach α ranging from .82 to .92. Single-factor and 7-factor confirmatory factor analysis showed satisfactory model-fit values. With one exception, all items demonstrated satisfactory loadings on their respective factors.The Swedish eHLQ demonstrated strong psychometric properties. It has the potential as a useful tool for a variety of purposes, including population surveys, intervention evaluations, and eHealth service implementations. Over the past decades, health care has undergone a rapid digital transformation. As health care services are increasingly delivered digitally, the internet has become a primary source of health information to people ,2. SwedeAdequate eHL allows people to increase the availability of health information, which improves their health knowledge and leads to positive outcomes, including a better understanding of the medical condition, increased empowerment, more effective self-management, and better communication with health care professionals -7. FurthIn 2006, Norman and Skinner  describeThe increasing use of eHealth services necessitates an effective instrument to assess health care consumers' eHL in areas of application, including population surveys, intervention evaluations, and eHealth service implementations . InsightThis study used both qualitative and quantitative approaches. We first translated and culturally adapted the Swedish eHLQ and then psychometrically tested the adapted instrument.The eHLQ contains 35 items on 7 scales representing the eHLF domains: using technology to process health information, understanding of health concepts and language, ability to actively engage with digital services, feel safe and in control, motivated to engage with digital services, access to digital services that work, and digital services that suit individual needs. Each scale consists of 4 to 6 items on a 4-point Likert scale ranging from “strongly disagree” to “strongly agree.” Scale scores range from 1 to 4, calculated on an index by averaging item scores within each scale with equal weighting. Each scale is presented separately, and no overall eHLQ score is calculated. Higher scores indicate higher abilities .License to translate the Danish version of the eHLQ to Swedish (02-2019) was obtained from Swinburne University, Australia. As required by the eHLQ developers, we used the translation integrity procedure (TIP) to maintain equivalence between the original (Danish) and translated (Swedish) versions of the instrument while ensuring the linguistic and cultural appropriateness of the Swedish version. The process was further facilitated by using clear “item intent” descriptions ,23.One of the eHLQ’s developers (Lars Kayser) chaired the TIP process. The translation and adaptation team included 2 native Swedish forward translators , 1 native Danish back translator (HG), a cognitive interviewer team , and 2 academic professionals , 1 fluent in both Swedish and Danish (MP) and both interested in local language and culture. All 3 translators had excellent Danish and Swedish language skills. The translation and cultural adaptation process involved three steps:(1) The original Danish eHLQ questionnaire was translated independently into Swedish by the 2 forward translators. The translators then used the item-intent descriptions, which thoroughly explained the intent of each item and scale, as a guide when synthesizing their translations. During the following team discussion, the best statements for each item were chosen and combined to form the first version of the Swedish eHLQ.(2) The first version of the Swedish eHLQ was back-translated by a native Danish-speaking translator, who had never seen the original version of the instrument. The Swedish-to-Danish back-translation was then compared with the Danish version of the eHLQ, and the items were discussed by the team to achieve consensus on the prefinal version of the Swedish eHLQ.(3) The prefinal Swedish eHLQ was then tested using cognitive interviews. Cognitive interviewing is valuable for ensuring accurate interpretations of items when translating and validating a questionnaire in another language and culture. This allows researchers to discover and correct items that are not interpreted as intended, thereby avoiding the future collection of inaccurate data. Cognitive interviewing does not require a large sample size, but the sample should represent a demographic variety ,24.Cognitive interviews were conducted with 9 adults (5 women) aged 18 to 80 (median 50) years, with varying educational backgrounds. The respondents were given a printed version of the questionnaire and were carefully observed while answering the items. The interviewer (AS) then went through each item with the individual respondents, focusing on items the respondent appeared to find difficult. The main questions were as follows: “What were you thinking about when you were answering that item?” and “Can you tell me why you selected that answer?” Participants were encouraged to elaborate on their interpretations of the items. A protocol was used for making notes during the interviews, which were also recorded, transcribed, and analyzed using a text summary .Results from the cognitive interviews revealed that although most items were understood as intended, minor revisions were needed to clarify a few items and instructions. The following corrections were made throughout the Swedish eHLQ:The Swedish term digitala hälsosystem  was consistently replaced by digitala vårdtjänster  in items 9, 13, 16, and 28.The Swedish phrase Jag är säker på… (I am confident that…) was replaced by Jag känner mig trygg att… (I feel safe that…) in item 1.When we reached an agreement on all formulations, the final version of the Swedish eHLQ was considered ready to be psychometrically tested.The data collection consisted of administering the Swedish eHLQ along with asking general questions about internet and eHealth service use. We also collected demographic data on participants’ age, sex, education, work situation, and health status. Because the topic of our study included digital use and literacy competencies, we chose to consistently administer paper-based questionnaires, although the eHLQ can also be administered on the web.Data were collected from 2 rural and 2 urban primary health care centers (PHCs) in northern Sweden. Receptionists were asked to hand out questionnaires to all adults (≥18 years) Swedish-speaking patients visiting the PHC for 2 weeks in November 2020. Participants could fill out the questionnaire at the PHC or at home, returning it using an attached stamped and addressed envelope. A total of 178 questionnaires were collected from PHC patients.An additional 64 questionnaires were filled out by parents of young children (<4 years) at the pediatric surgery or neonatal department of a hospital in southern Sweden. Nurses in the participating departments handed out questionnaires to Swedish-speaking parents of in-patient children in the spring of 2021. Otherwise, the procedure was identical to the PHC data collection.Data analysis was performed using SPSS version 25) and JAMOVI (version 2.2.3). Cases with ≥50% (n=18) missing values were excluded (n=2). Other missing values were replaced using the expectation––maximization algorithm imputation in SPSS. Demographic characteristics were reported by frequency and percentage (categoric variables) or mean and SD (continuous variables). Floor and ceiling effects were considered present if >15% (n=35) of participants reported the lowest or the highest response option for an item [ and JAMOBecause the aim of this study included the validation of a Swedish version of an instrument with scales that were defined a priori, our analyses were confirmatory. To evaluate internal consistency, Cronbach α was calculated separately for each scale. Internal consistency was considered good when Cronbach α was .70-.95 . We alsoEthical approval was granted by The Regional Ethical Review Board at Umeå University (no: 2014-179-31M) and The Swedish Ethical Review Authority (no. 2019-0341) and included a complementary application regarding expanded data collection. All steps were managed according to the General Data Protection Regulation and the ethical principles described in the Helsinki declaration .A total of 236 individuals completed the questionnaire, of whom 172 (73%) were PHC patients, and 64 (27%) were parents of hospitalized children. The sample included 129 (55%) women and 105 (45%) men aged 20-93  years; 130 (55%) worked, and 112 (48%) had a university education see .The eHLQ mean scores ranged from 2.58 (SD 0.73) on scale 7  to 3.04 (SD 0.55) on scale 2 . As with the single-factor CFA, all factor loadings were satisfactory to high except for item 3 on scale 6  to 0.99 . The second highest interfactor correlation coefficient, 0.97, was between subscales 6 and 7 see .This study translated, culturally adapted, and evaluated the psychometric properties of the Swedish eHLQ. We used a systematic and rigorous translation and cultural adaptation process to reproduce the original instrument’s concepts and meanings. Our data from a diverse validation sample demonstrated that the Swedish eHLQ has strong psychometric properties and is in line with the psychometric outcomes of the versions in Danish and other languages ,21,33.As recommended for examining the validity of a translated instrument, this study used qualitative and quantitative approaches. During the first phase, we adopted TIP, which includes a multistep translation and review process and detailed item intent descriptions ,23. ResuThe proportion of unanswered items varied from 0.8% to 4%, indicating an acceptable item-response rate and a good understanding of the items. Our data revealed a few items with floor effects, but most items showed ceiling effects, which might indicate that a large proportion of our sample had confidence and trust in their abilities and the eHealth system . A problAll scales demonstrated good internal consistency, with a Cronbach α of >.80. Consistent with Danish and Australian eHLQ validation studies, scales 2 and 6 showed the lowest values ,21. Our Given our results, the Swedish eHLQ can be considered a good replication of the original instrument. The multidimensionality of this eHL instrument adds to the health literacy and eHL instruments currently available in Sweden. Although previous instruments such as the eHEALS have focused on individuals’ competencies, the eHLQ has the added perspective of the interaction between the individual and the eHealth systems ,20. TherData collection was conducted during the first year of the COVID-19 pandemic, which created a considerable workload burden and a need among PHC and hospital staff to prioritize their tasks. The reduced number of PHCs able to participate and the shortened time available for data collection also reduced the intended sample size. Nevertheless, we decided our sample of 236 participants was appropriate, especially since the original eHLQ is a thoroughly designed and well-researched questionnaire ,36. UsinOur psychometric testing sample, which included primary health care patients and parents of in-patient children, represented a diversity of educational levels, genders, ages, and work statuses. Therefore, although it might not represent the national average, it reflected a generally diverse sample.Another limitation is that eHealth–literate individuals might be overrepresented in this study because they are more likely to participate than people who consider themselves to have poor eHealth skills or who have negative attitudes toward eHealth service usage. This phenomenon is, however, difficult to avoid.This study suggests that the Swedish eHLQ is a reliable instrument, with good linguistic equivalence to the original and robust psychometric properties. Our sample consisted of PHC patients and parents of hospitalized children, but the questionnaire should be further tested in different demographic and disease groups. We expect the Swedish eHLQ will be a valuable tool for assessing multidimensional perspectives of eHL when conducting population surveys, intervention evaluations, and eHealth service implementations.


Under California Senate Bill 493, pharmacists can order patient laboratory tests (labs). Currently, it is unknown if this service affects patient outcomes or pharmacy operations. Does lab ordering by pharmacists improve access to care, improve quality outcomes, and/or affect pharmacy operational functions?A 13-month pilot study was conducted at 2 Kaiser Permanente (KP) outpatient pharmacies where pharmacists provided extended adherence consultations and ordered hemoglobin A1c (HbA1c) labs for patients nonadherent to their oral diabetic medications with an HbA1c ≥ 8% or missing annual labs. Clinical outcomes of the pilot study were compared with a similar patient population at KP who concurrently received lab orders from their primary providers, defined here as the “usual care model.”P = 0.10). A significantly greater proportion of patients in the pharmacist group achieved an HbA1c < 8% within the follow-up period of 30-180 days (34.9%), compared with the usual care group . Of the patients who completed labs during the evaluation period, 38 (43.7%) patients in the pharmacist group and 111 (15.7%) patients in the usual care group had prelaboratory values ≥ 8% within the previous 12 months. The average pre-HbA1c value was 9.47% in the pharmacist group, and the average post-HbA1c value was 8.68% (P < 0.01). For the usual care group, the average pre-HbA1c value was 9.70%, and the average post-HbA1c value was 9.43% (P = 0.06). When comparing the difference in HbA1c reduction between the 2 groups, there was a larger decrease in HbA1c in the pharmacist group, but this difference was not significantly different (P = 0.06). The pilot study added an average of 5 minutes per patient encounter to the pharmacy workflow but did not affect overall patient wait times for receiving outpatient prescriptions.OBSERVATION: Of the 793 HbA1c lab orders, 87 (11.0%) were generated by a pharmacist, and 706 (89.0%) were generated by the usual care group. Forty-three (49.4%) patients in the pharmacist group completed their labs compared with 279 (39.5%) patients in the usual care group (Laboratory ordering by pharmacists in the outpatient setting improved access to care, improved quality outcomes, and did not adversely affect pharmacy operations. What is already known about this subjectCalifornia Senate Bill 493, passed in 2013, allows pharmacists to order and interpret laboratory tests (labs), furnish birth control and smoking cessation products, and administer a wide variety of vaccines without a physician protocol for patients aged 3 years and over.Based on the language in California Assembly Bill 1114, it is uncertain how pharmacists will be reimbursed for providing lab ordering services.The lack of access to medical records in the community pharmacy setting may be a barrier to implementation of various services recognized by Senate Bill 493.What this study addsPharmacists are able to positively affect clinical outcomes for patients through lab ordering in the outpatient pharmacy setting.Lab ordering by pharmacists in the outpatient pharmacy could be operationally sustainable, but potential barriers, such as volume of lab orders and a lack of reimbursement, may limit this service in traditional independent and chain pharmacies.1 Through this expanded scope of practice, pharmacists are able to order and interpret labs, furnish birth control and smoking cessation products, and administer a wide variety of vaccines without a physician protocol for patients aged over 3 years. However, only a minority of pharmacists participate in these services. A study by Gomez (2017) identified that only 11% of community-based, retail pharmacies offered pharmacist-prescribed hormonal contraception.2 Rodriguez et al. (2016) looked at barriers to implementing pharmacist-furnished hormonal contraception and identified a lack of pharmacy staff, concerns about liability, and the need for additional training as some of the most common factors that impeded pharmacist participation.3Pharmacists in California have had an expanded scope of practice since the passing of California Senate Bill (SB) 493 in 2013.6 a search of the literature rendered limited information on laboratory test (lab) ordering services and their relationship with outpatient operations or clinical patient outcomes. This is likely because of the difficulty that independent and community pharmacies have in accessing patient medical records, which makes this type of study challenging. Moreover, a bill does not currently exist in California that would provide reimbursement for lab ordering services provided by pharmacists, potentially hindering them from offering the service altogether. At Kaiser Permanente (KP), pharmacists in the outpatient setting have access to the electronic medical record (EMR), KP HealthConnect, to order and review patient labs. However, even within an integrated system such as KP, where improving patient medication use outcomes are integrated into the workflow, incremental resources are needed to accommodate for pharmacists’ time in providing these services. While allowing pharmacists to work at the top of their license would increase access to care, reduce care gaps, and improve quality outcomes,8 these outcomes have yet to be studied with regards to lab ordering in the outpatient setting. Our pilot program aimed to implement outpatient pharmacist lab ordering coupled with face-to-face consultations for patients nonadherent to their oral diabetes medications with a hemoglobin A1c (HbA1c) ≥ 8% or a missing annual HbA1c. Our objectives were to report on the operational effect of the program and assess its effect on clinical patient outcomes as measured by HbA1c goals.While there are several studies investigating pharmacist perceptions or economic evaluations of SB 493 implementation,9 MPR is defined as the percentage of the total days supply for all fills of a given drug in a particular time period divided by the number of days in the time period.10The pilot study was conducted at 2 sites from October 1, 2016 through October 31, 2017—KP Los Angeles Medical Center 4900 Sunset Pharmacy and KP Baldwin Park 24-Hour Pharmacy. Ten pharmacists engaged in this pilot and were required to complete a 60-minute online training for diabetes competency and a 60-minute live online session on lab ordering and results follow-up in KP HealthConnect. Patients included in this study belonged to a KP outpatient pharmacy clinical service (OPCS) program that targets patients with diabetes who have suboptimal rates of oral antidiabetic medication adherence, as measured by a medication possession ratio (MPR) of < 80% and an HbA1c lab result ≥ 8%, or missing annual HbA1c.When a patient in the OPCS program was identified during prescription processing for any medication in the pharmacy, a care management summary sheet was generated, displaying the most recent labs for HbA1c, low-density lipoprotein cholesterol, and a list of the 20 most recent prescription fills within the previous 12 months. In addition, the summary sheet displayed the respective MPRs and days supply remaining for the patient’s chronic medications. At the time that patients picked up any requested medication, the pharmacist provided an extended face-to-face consultation to identify and address barriers to adherence, along with an explanation of what an HbA1c result signified and its respective goals for patients with diabetes. In this “usual care model,” the primary care provider then ordered a monitoring or screening HbA1c lab for patients who agreed to restart their nonadherent medications as prescribed. To assess outcomes, patients had to receive OPCS consultations and complete HbA1c lab orders placed by their providers within 30 to 180 days after their consultations.With the new workflow designed to implement the expanded scope of practice given to pharmacists under SB 493, pharmacists in the pilot program ordered the monitoring lab at the time of the face-to-face extended consultation for patients who agreed to restart an oral diabetes medication rather than request an order from the provider. This bypassed the need for a potential office visit or provider contact to order such labs. Four weeks after patients restarted their medications, they could visit any KP laboratory for the lab test. The results were then routed electronically through KP HealthConnect to the pharmacists, where they could electronically connect with patients’ providers to determine the need for follow-up and potential medication dosage change. Patients were followed from day 30 to day 180 after the lab orders were placed. Patients who did not complete labs during the observation period were excluded in the analysis for HbA1c outcomes.To assess operational effects and job satisfaction of pharmacists involved in this pilot program, a survey consisting of seven 5-level Likert scale questions was conducted at the pilot sites. The pharmacy manager at each pharmacy collected the surveys from the participating pharmacists and submitted the results to the pilot program’s coinvestigators. Also, an interview was conducted with one of the pharmacists who participated in the pilot program.The primary goal was to determine if OPCS lab ordering by pharmacists improved access to care, as measured by the number of completed labs ordered by a pharmacist. In addition, this pilot program aimed to look at how this expanded role of outpatient pharmacists could improve quality outcomes, as measured by HbA1c. This study also looked at the pilot program’s effect on outpatient pharmacy operational functions and the satisfaction of pharmacists participating in the program.11 Lab ordering by pharmacists was the only independent variable tested in this study.The coinvestigators performed a clinical review process through a protocol approved by the KP Southern California Institutional Review Board. The process involved retrospective chart reviews for the following patient-specific information: age, sex, self-reported ethnicity and race, date of lab order and completion, laboratory result, and prelaboratory date and value within the previous 12 months, if applicable. An HbA1c value < 8% was the target outcome for all patients, regardless of age or comorbidities.P ≤ 0.05.Continuous variables were compared between groups using an unpaired 2-tailed t-test and within groups using a paired 2-tailed t-test. Categorical variables were analyzed for statistically significant differences between groups using the chi-square test with Yates correction. Statistical significance was defined using P > 0.05 for all reported races). The average age was higher in the pharmacist group at 61 years compared with 58 years for the usual care group. This finding was statistically significant (P = 0.04).During the study period , 793 patients were included. P = 0.10). A significantly greater proportion of patients in the pharmacist group achieved an HbA1c < 8% (34.9%), compared with the usual care group . Of the patients who completed labs during the evaluation period, 38 (43.7%) patients in the pharmacist group and 111 (15.7%) patients in the usual care group had prelaboratory values ≥ 8% within the previous 12 months (P < 0.01).Of the 793 HbA1c lab orders, 87 (11.0%) were generated by a pharmacist, whereas 706 (89.0%) lab orders were generated by the usual care group . Forty-tP < 0.01). For the usual care group, the average pre-HbA1c was 9.70% and the average post-HbA1c was 9.43%, with an average decrease of 0.27 percentage points. This difference was not statistically significant (P = 0.06). When comparing the difference in HbA1c reduction between the 2 groups, there was a larger decrease in the pharmacist group, but this difference was not significantly different from the usual care group (P = 0.06). The average time lapse between the date the lab order was placed and the post-HbA1c lab draw was 82 days for the pharmacist group and 79 days for the usual care group. This difference between the 2 groups was not statistically significant (P = 0.43).The pharmacist group had an average pre-HbA1c value of 9.47% and an average post-HbA1c value of 8.68%, with an average decrease of 0.79 percentage points, which was found to be statistically significant  services to these patients. MTM does not involve the ordering of labs, but studies have shown that MTM services, including restarting nonadherent medications, can improve clinical outcomes. Pinto et al. (2014) demonstrated that MTM services after 1 year decreased patient HbA1c by 0.27 percentage points.1 ordering and interpreting laboratory tests was likely not included in the pharmacy education curriculum. Moreover, because lab ordering in the outpatient setting has only been implemented in a pilot setting, new KP employees are not trained on lab ordering as part of the onboarding process.Interestingly, the survey and the interview revealed that pharmacists prefer hands-on training with regards to lab ordering. Since SB 493 came to fruition in 2014,13 With the passing of California Assembly Bill 1114 in 2016, pharmacist services including immunizations, smoking cessation counseling, and furnishing of travel medication, hormonal contraception, and nicotine replacement therapy will be reimbursed through Medi-Cal by July 1, 2021.14 While this bill provides an outlet for pharmacists to be reimbursed for their services in the outpatient setting, it is uncertain, based on the language of the bill, how lab ordering services will be reimbursed.Outpatient pharmacists in the community have had challenges implementing lab ordering into their practice for several reasons. Pharmacies would require access to patient medical records. For KP, the integrated medical system facilitates lab ordering and the access of results through the synced EMR. Moreover, O’Connor et al. (2012) identified barriers to implementing pharmacogenetic lab ordering in the outpatient pharmacy setting that included the lack of insurances recognizing pharmacists as health care providers, which would prevent reimbursement and even ordering of such labs.The 5-minute encounter time did not affect patient wait times in the pharmacy, but it did add a workflow step for the pharmacists. The lack of a payment model for lab ordering by pharmacists creates a barrier for implementation within outpatient pharmacies. While KP does not rely on reimbursement for individual pharmacist-provided services, if lab ordering were to increase in volume and scaled up to include other lab tests, incremental resources to meet the changing workflow needs would be necessary.Pharmacist intervention through outpatient lab ordering showed a positive effect on improved lab draw adherence and HbA1c reduction. However, these outcomes may be influenced by several confounding factors, including patient age, sex, race, and comorbidities. Furthermore, while the pharmacist intervention significantly reduced HbA1c values, the average post-HbA1c was still ≥ 8%. Additional pharmacist services may be necessary to further reduce HbA1c values in order to achieve clinical targets. Limitations of the pilot study include the smaller size of the pharmacist group compared with the usual care group and the large proportion of patients in both groups who did not complete ordered labs. Future randomized studies will be necessary to address differences in group size and possible confounding variables, as well as reasons for low completion rates of ordered labs.In conclusion, engaging pharmacists in lab ordering in the outpatient pharmacy setting improves access to care, enhances quality outcomes, and does not adversely affect pharmacy operations. Pharmacists who participated in this pilot program had a positive experience and would benefit from the opportunity to provide lab ordering services to patients.


Prunus domestica subsp. insititia). The test plantlets were cultivated under a Philips GreenPower LEDs research module illumination system with four spectral regions: white (W), red (R), blue (B) and mixed (W:R:B:far-red = 1:1:1:1). The control plantlets were cultivated under fluorescent lamps (FL) and the photosynthetic photon flux density (PPFD) of all treatments was set at 87 ± 7.5 μmol m−2 s−1. The effect of light source on the selected physiological, biochemical and growth parameters of plantlets was monitored. Additionally, microscopic observations of leaf anatomy, leaf morphometric parameters and stomata characteristics were carried out. The results showed that the multiplication index (MI) varied from 8.3 (B) to 16.3 (R). The MI of plantlets grown under mixed light (WBR) was 9, lower compared to the control (FL) and white light (W), being 12.7 and 10.7, respectively. In addition, a mixed light (WBR) favored plantlets’ stem growth and biomass accumulation at the multiplication stage. Considering these three indicators, we could conclude that under the mixed light, the microplants were of better quality and therefore mixed light (WBR) was more suitable during the multiplication phase. A reduction in both net photosynthesis rate and stomatal conductance in the leaves of plants grown under B were observed. The quantum yield (Yield = FV/FM), which represents the potential photochemical activity of PS II, ranged from 0.805 to 0.831 and corresponded to the typical photochemical activity (0.750–0.830) in the leaves of unstressed healthy plants. The red light had a beneficial effect on the rooting of plum plants; the rooting was over 98%, significantly higher than for the control  and the mixed light . In conclusion, the mixed light (WBR) turned out to be the best choice during the multiplication phase and the red LED light was more suitable during the rooting stage.In recent years, light emitting diodes (LEDs), due to their low energy consumption, low heat emission and specific wavelength irradiation, have become an alternative to fluorescent lamps (FLs) in plant tissue culture. The aim of this study was to investigate the effects of various LED light sources on the in vitro growth and rooting of plum rootstock Saint Julien ( Prunus domestica L.) is a very important fruit species in the temperate climate zone with a broad distribution in the southern parts of Europe, but it is also well adapted to the climate in its northern parts. The plum fruits are consumed fresh, dried, canned or are used for brandy making. Plums provide vitamins, minerals and antioxidants, such as flavonoids, carotenoids and glutathione. They are excellent functional foods for cardiovascular health due to their high fiber and potassium content and cholesterol-reducing capacity [Prunus domestica subsp. insititia) is one of the most preferred plum culture clonal rootstocks in Europe [The European plum .Numerous studies reported successful applications of LEDs in promoting in vitro growth and morphogenesis in various plant species . Better Populus  and CastCastanea  but the a [Pinus ,29, coffa [Pinus , Eucalyprophylla , Cedrelafissilis , Pinus sii-regis  have maiii-regis . The stiii-regis . There aii-regis . The aimThere were significant differences in the appearance of micropropagated plum plantlets when different light sources were applied , Table 1The plantlets grown under red LED light (R) and those grown under mixed LED light (WBR) had the greatest average stem length, 16.5 mm and 15.1 mm, respectively, although a statistically significant difference with the control (FL) was found only in response to red light treatment . The plaIn the studied plum plantlets, the multiplication index (MI) varied in a wide range—from 8.3 (B) to 16.3 (R)—with the highest value reported in plants grown under red light . Under ta and total chlorophyll content  had a positive effect on the photosynthetic pigments because the microplants cultivated under blue light showed the highest chlorophyll  content . The hig content . The dat−1 FW for plants cultivated under FL and WBR, respectively. In the present experiment, the quality of light had a significant effect on the total phenolic content and antioxidant activity in the plantlets . The totp < 0.05).The values of WBR total antioxidant capacities expressed either as % discoloration of DPPH radical reagent or as Trolox equivalent (TE) were twofold higher, 8.59 mg TE/100 mL and 44.09, than the values for FL which were 4.54 and 21.18, respectively. The analyses also showed a correlation between the total phenolics content and the antioxidant activity (2/mmol H2O); in all the other treatments, it was comparable and varied in a range within 65–75% compared to the control.A reduction in both net photosynthesis rate and stomatal conductance in the leaves of plants grown under B were observed . The tra0 to FM level with distinct J and I phase , maximum (FM) and variable (FV) fluorescence of the control plantlets and plantlets grown under LED light did not differ significantly (V/FM), which represents the potential photochemical activity of PS II, ranged from 0.805 to 0.831 and corresponded to the typical photochemical activity (0.750–0.830) of the leaves of unstressed healthy plants [V/FM) was reported for the leaves of the plants under mixed light (WBR) and there was a statistically significant difference to the control plant’s yield (FL). The minimum  can be used for the assessment of the PSII state and functional activity in relation to the amount of absorbed energy [abs was observed in plants cultivated under mixed and blue LEDs, followed by plants from FL and W light treatments. The lowest PIabs was estimated for plants under red light. The performance index  was marked in the plantlets cultivated under mixed light (WBR) and the lowest was marked in the plantlets cultivated under red light  had the thickest leaf lamina. Similar leaf lamina thickness was observed for the leaves under mixed blue and red light (WBR). Between these two treatments, there was no significant difference but their morphological parameter values were much higher than those of the other three variants. The thinnest lamina was observed in the leaves formed under monochromatic red light (R). A similar result was observed for the mesophyll, since the thickest mesophyll was seen in leaves under white light (W), followed by the leaves under mixed light (WBR). The monochromatic blue light (B) led to the thinnest mesophyll. The highest values for the palisade and the spongy parenchyma were measured in leaves differentiated under white light (W), followed by the leaves of plantlets grown in mixed light (WBR). The only significant difference was estimated for the palisade parenchyma between W and WBR light treatments. The lowest values of the palisade parenchyma were found in the leaves under blue light (B) but they were similar to the other light treatments, red light (R) and fluorescent light (FL), and furthermore no significant difference was noted. The mixed light (WBR) increased the thickness of both adaxial and abaxial epidermises, although the difference for the adaxial epidermis was statistically significant only in FL and R. Red light (R) has the opposite effect, as the adaxial and abaxial epidermises were thinner .In the examined leaves from all light treatments, the stomata had elliptical or round shapes . The larLight had a significant effect on the rooting of in vitro cultivated plantlets of ‘St. Julien’ plum rootstock . Under wGossypium hirsutum, Anthurium andreanum, Brassica napus, Musa acuminata, etc. [Numerous studies have reported the applications of LEDs and made a comparison with the white FL regarding the possible effects on promoting the in vitro organogenesis, growth and morphogenesis of various plant species such as ta, etc. ,22,39,40The possibility to modulate the light spectrum in accordance with plant demands appears to be one of the most important advantages of the LED techniques . It is kv/FM), although the difference to B was not statistically significant. Considering these indicators, we could conclude that under the mixed light, the microplants were of better quality. Furthermore, the size of the plum explants allows for easier handling and manipulation, which can speed up the process of micropropagation and has a significant practical value. Therefore, we can conclude that mixed light (WBR) is more suitable during the multiplication phase.The plantlets from plum rootstock ‘St Julien’, used in the current study, were cultivated under various light sources. The multiplication index (MI) is one of the most important indicators in plant micropropagation, but it should be considered in a complex manner with other indicators. Under mixed LED light (WBR), the plantlets’ leaf number and size, photosynthetic pigment content and net photosynthetic rate  did not Rubus idaeus L.) [Vaccinium corymbosum L.) [Pyrus communis L. [Lilium sp. [Chrysanthemum sp. [Doritaenopsis sp. [Lycium barbarum L. [Our previous research with raspberry (aeus L.) , highbusosum L.)  and Pyrumunis L.  showed tmunis L.  who obselium sp. , Chrysanemum sp. , Doritaepsis sp.  and Lycibarum L. . Mentha and Fragaria [Other researchers have shown that the combination of red and blue LEDs had enhanced the growth of plants from the genera Fragaria ,19,49. MFragaria  also nota fluorescence is another indicator of the functional activity of the photosynthetic apparatus of plants. The chlorophyll a fluorescence induction has been thoroughly studied since 1931 when Kautsky and Hirsch discovered the negative correlation between the fluorescence intensity and carbon dioxide fixation [a fluorescence in the leaves of plants cultured in a controlled environment could be affected by the light [total) presents not only the functional activity of the PSII, but also the PSI along with the rate of electron transport chain between them [total is closely related to the overall growth rate and survival of plants under stress conditions and has been described as a very sensitive and reliable parameter in the JIP test. The highest value of PI total was recorded in the plantlets cultivated under mixed light (WBR) and this accurately corresponded to the highest value of biomass accumulation in plantlets  and stomatal conductance (gs). One possible explanation could be related to the fact that the measurements were performed on physiologically older leaves than the first fully developed leaves in the other variants in which the fluorescence was evaluated.The increased values of ψPlum is a plant genetically predisposed towards accumulating secondary metabolites of the phenolic group. The phenolic compounds are well known for their antioxidant properties and their synthesis can be stimulated by various environmental factors . The in 2•−), hydroxyl radical (OH•) and peroxy radical (ROO•). The free radicals can cause protein denaturation, lipid peroxidation and oxidative DNA damages and negatively affect membrane fluidity [It is known that light that affects plant morphogenesis and metabolism is one of the factors responsible for the production of reactive free radicals, such as superoxide anion (Ofluidity . Antioxifluidity  have sim−2 s−1) could be effective either for modifying the potential of Gerbera jamesonii Bolus shoot multiplication, or for controlling the plant morphometry and photosynthetic pigment content [Brassica napus in in vitro cultures was higher under blue light than under white light [The studies on gerbera showed that the combination of red (70%) and blue (30%) light with specific light intensity  and mixed light (WBR) plants. These results are similar to the previous observations, which showed that the red light stimulated raspberry shoot elongation at the multiplication stage . In addiOncidium , blueberOncidium , Scrophusimensis , stevia simensis  and Carpte Maxim .According to Manivannan et al. , the stiSpathiphyllum cannifolium [Mirtus communis [Vanilla planifolia. The same decrease was found by Martínez-Estrada et al. [Anthurium andreanum and Lotfi et al. [Pyrus communis. As noted earlier, some authors agreed on the positive role of red light, and high R:FR light ratio on shoot proliferation . In addinifolium  and Mirtcommunis . On the communis  observeda et al.  who studi et al.  who studThe main effects of the R light are explained by its effect on the phytochrome and the synthesis of cytokinins in the plant tissues . The cytAmelanchier alnifolia leaves compared with those developed under FL light [2 absorption [Predictably, the thickest mesophyll was measured in plum leaves formed under white light (W), followed by the leaves of mixed light (WBR), while the leaves that developed under monochrome light were significantly thinner. That observation confirmed the fact that the full light spectrum was beneficial during leaf ontogeny. Additionally, in pepper plantlets and cherry tomato plants, the leaf thickness, and the palisade parenchyma thickness, respectively, were high in leaves developed under RB light ,79. The FL light . StudyinFL light  found thFL light . It is psorption  and mighsorption .2, and can affect the transpiration and nutrient uptake [According to a number of authors, the blue light is necessary for a proper stomatal opening, can improve the access to COt uptake ,86,87,88The blue and red spectra are required for chlorophyll synthesis and foliar growth and their combination in a suitable proportion is important for overall plant growth and development . IncreasRooting is an important step in whole plant formation during the micropropagation process and along with the shoot induction has often been evaluated. As aforementioned, the light had a significant effect on the rooting of in vitro cultivated plantlets from the ‘St. Julien’ plum rootstock . Under tChrysanthemum morifolium cv. ‘Ellen’. The red light also stimulated the growth of adventitious roots in Morinda citrifolia [Panax ginseng. The data of Shulgina et al. [Stevia rebaudiana Bertoni shoots, but stimulated root system development.Different reports have indicated that the R light alone is effective in root induction, but the effects of different light qualities on root development are often contradictory in the available literature. Kurilčik et al.  reportedtrifolia  and, acctrifolia , R lighta et al.  showed tPrunus domestica subsp. insititia). The experiment was carried out with plum rootstock ‘Saint Julien’  were taken in mid-April 2019 from young shoots (8–10 cm); their leaves were removed and surface sterilized by immersion in 70% ethanol for 30 s and in a 5% solution of calcium hypochlorite containing 2–3 drops of Tween for 10 min. The shoots were then rinsed four times (10 min each) in sterile distilled water. Nodal segments (10 mm) were cut from the shoots and inoculated on the solid MS medium [−1 sucrose and 6.5 g L−1 Phyto agar . The medium was adjusted to pH 5.6 before autoclaving at 121 °C for 20 min. The in vitro shoot culture was maintained on the above-mentioned nutrient medium at 4-week subculture intervals. Plantlets were placed in baby food glass jars  with transparent Magenta B-Cap lids with 25 mL nutrient medium per vessel. In each vessel, five explants were incubated. The cultures were cultivated at 22 ± 2 °C under a 16 h photoperiod , provided using a Philips GreenPower LED research module . Four groups of LEDs emitting in white (W), red , blue  and far red  were used. Plantlets grown in the same conditions but under fluorescent lamps (FL) served as the control. Shoots of S medium , supplemEach multiplication experiment used six jars (with five explants in it) for each light treatment and the experiment was repeated twice. −1 sucrose and 6.5 g L−1 Phytoagar, pH 5.6. Plantlets were grown in baby food glass jars at the corresponding light regimes and temperatures described above. Data on the rooting percentage, number of roots per rooted microcutting and the length of roots were recorded three weeks after the beginning of the experiment. Rooting experiments were performed on 30 shoots per light treatment (six jars × five explants) and were repeated twice. Apical shoots (10–15 mm long) with two to three leaves from the previous experiment with shoot multiplication were transferred to the rooting medium containing half-strength MS macronutrients, full-strength micronutrients and vitamins, 1.5 μM IBA, 20 g LThe plants were cultivated for four weeks until their transfer to the fresh culture medium. The analyses were performed after five consecutive passages in total and the following parameters estimating the plants development were measured: fresh (FW) and dry biomass weight (DW), length of the shoots, content of the photosynthetic pigments, total polyphenols, antiradical activity, gas exchange rate and chlorophyll a fluorescence.The base of the plants was washed with running water to remove traces of the nutrient medium, then dried with filter paper and the fresh weight (FW) of the plants was measured. The dry mass (DW) of the plants was determined after drying at 105 °C (±5 °C) to a constant mass.A morphological observation of leaf anatomy was conducted. Multiplication index (MI) was calculated as the number of proliferated shoots from one explant. −2 s−1 PPFD and an ambient CO2 concentration of 1100 vpm. Each plantlet was placed in the conifer measurement chamber, and the base of the shoot was wrapped in wet filter paper to prevent drying. The net photosynthesis rate , transpiration intensity , photosynthetic water use efficiency  and stomatal conductance  were determined.A gas exchange analysis was performed with a portable gas exchange system LCpro+  at a light intensity of about 130 µmol ma fluorescence induction curves (OJIP) were recorded on each fully developed youngest leaf of five representative plants with a Plant Efficiency Analyser . The analyzed leaves’ spot areas were adapted to dark with special clips for 40 min. The induction curves of the rapid chlorophyll a fluorescence (OJIP test) were recorded after illumination with 3000 µmol m−2 s−1 PPFD for 1 s. The primary data were processed with the PEA Plus Software . The parameters of the OJIP test (Chlorophyll JIP test  were intn = 12). After the filtration of the extract, 1 mL was mixed with 2 mL acetone, and chlorophyll-a (Chl a), chlorophyll-b (Chl b) and carotenoid (Car) contents (µg g−1 FW) were spectrophotometrically determined at absorbances of 663 nm, 645 nm and 470 nm, respectively, according to [Photosynthetic pigments were extracted from fully developed leaves using 0.1 g of fresh plant material in 10 mL of 80% aqueous acetone  using a standard curve and were presented as mg g−1 FW. The standard curve was prepared with gallic acid  in the range 0–500 mg L−1. The total amount of polyphenol compounds in the plant extracts was determined with the Folin–Ciocalteu reagent , accordi−5 mol L−1 DPPH (0.06 µmol). The measurement of extract absorption was performed twice: first, immediately after mixing the components (0 min); second, after 30 min at 515 nm and when a parallel blank sample that contained only distilled water was used. The antiradical activity was expressed as % discoloration according to Equation (1): Antioxidant activity was measured in the extracts obtained for the total phenolics and 2,2-diphenyl-1-picrylhydrazyl (DPPH) . The inc2 = 0.9858). The Trolox was used as a standard.The DPPH radical scavenging activity of the sample was also expressed as mg Trolox equivalent antioxidant capacity (TEAC) by the formula obtained from the standard curve (y = −0.1954x + 0.708R2) from the middle part of the leaf lamina were fixed in 3% (m/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 12 h at 4 °C. Several transverse handmade sections mounted on slides with glycerol were used as materials for the histological study. The observations were carried out with Amplival 4 light microscope . An average number of fifteen microphotographs with high resolution (2560 × 1960 pixels) were taken with the EcoBlue digital microscope (EC.1657), with an integrated 5.0 MP USB-2 camera  with 400× magnification. The microphotographs were used also for the leaf’s anatomy observation and for the measurements of the lamina (LL), mesophyll (M), palisade parenchyma (PP), spongy parenchyma (SP), and both the adaxial (AdE) and abaxial (AbE) epidermis. Dimensions were presented in µm according to the recommendations of the ImageJ . The stomata observations were performed by using small fragments from the central parts of five leaves, which were preliminary rinsed with distilled water, bleached and rinsed again. The prepared samples were placed on microscopic slides and covered with glycerol. The estimation of the length and width (in µm) of the stomata, as well as the stomatal distribution (number/mm2), was based on 30 measurements performed with an Amplival 4 light microscope  for each light treatment.Anatomical observations, for each light treatment, were performed on twenty leaves taken from the 3rd or 4th nodes of the in vitro plants. Small segments , followed by a comparison of group means (Tukey’s b test) with the program SPSS ver. 26.0 (IBM).Prunus domestica subsp. insititia ‘Saint Julien’). The plantlets exhibited specific growth needs during the different stages of their micropropagation. The results from the current study showed that the plantlets cultivated under the mixed LED light (WBR) at the multiplication stage had the highest fresh (FW) and dry (DW) biomass in comparison to the control plants and to the other light sources. In addition, there was a trend for greater stem length under the mixed light (WBR). Considering these three indicators, we could conclude that under the mixed light, the microplants were of better quality and therefore mixed light (WBR) was more suitable during the multiplication phase. The red LED light stimulated the rooting. The current study protocol can be considered as an effective and affordable method for the large-scale propagation of valuable plum genotypes, which is also suitable for commercial purposes. Furthermore, it can be applied in future research aiming for the successful in vitro cultivation of other ‘difficult-to-propagate’ Prunus species or other woody plants.In the present study, LED sources with a different light spectra distinctively affected the growth and development of in vitro cultivated plum plantlets (


The microenvironment and stem cell fate guidance of post‐traumatic articular cartilage regeneration is primarily the focus of cartilage tissue engineering. In articular cartilage, stem cells are characterized by overlapping lineages and uneven effectiveness. Within the first 12 weeks after trauma, the articular inflammatory microenvironment (AIME) plays a decisive role in determining the fate of stem cells and cartilage. The development of fibrocartilage and osteophyte hyperplasia is an adverse outcome of chronic inflammation, which results from an imbalance in the AIME during the cartilage tissue repair process. In this review, the sources for the different types of stem cells and their fate are summarized. The main pathophysiological events that occur within the AIME as well as their protagonists are also discussed. Additionally, regulatory strategies that may guide the fate of stem cells within the AIME are proposed. Finally, strategies that provide insight into AIME pathophysiology are discussed and the design of new materials that match the post‐traumatic progress of AIME pathophysiology in a spatial and temporal manner is guided. Thus, by regulating an appropriately modified inflammatory microenvironment, efficient stem cell‐mediated tissue repair may be achieved. Stem cells recruited at the defect site are characterized by overlapping lineages and uneven effectiveness. The inflammatory microenvironment plays a decisive role in guiding the fate of stem cells. Design strategies along with the evolution of inflammatory microenvironment are summarized and effective drug strategies are highlighted. Moreover, a design trend for new generation of tissue engineering repair materials is proposed. It is particularly difficult for articular cartilage within load‐bearing joints to recover normal function following injury, as disturbances of the articular inflammatory microenvironment (AIME) result in incomplete cartilage repair. In fact, AIME reconstruction determines the repair outcome within 3 months after injury, while the evolution of the AIME is associated with a systemic inflammatory response and a cytokine storm.Articular cartilage injury is an important problem worldwide.1 Arti The strategy of creating cartilage through tissue engineering using cells and exogenous stimuli has progressed with respect to articular cartilage repair. This is one of the most promising methods for treating articular cartilage injury. Moreover, stem cell‐based tissue engineering offers a new therapeutic approach for patients.Currently, only surgery and tissue engineering regeneration can repair damaged cartilage.6 The  However, the mesenchymal stem cells (MSCs) used for these two methods are heterogenous, and the overall effectiveness of stem cells and the problem of overlapping lineages contribute to the uncertainty of tissue repair. In this review, we discuss the types of stem cells that contribute to articular cartilage repair and provide an overview of their fate. Subsequently, we describe the changes that occur in the microenvironment of the injured cartilage as inflammation develops and the effects of these changes on stem cell regeneration. We believe that the AIME plays a decisive role in the fate of stem cells and identify the key factors that determine the fate of stem cells at each time point during cartilage repair. The goal is for endogenous or exogenous stem cells to differentiate into cartilage by regulating the AIME, maximizing the potential of their fates, and achieve complete repair. Finally, we discuss the opportunities and challenges arising from the use of AIME deconstruction technologies, which will enable us to design a new generation of tissue engineering materials.Two types of stem cells are used in tissue engineering: endogenous stem cells recruited from in situ tissue and exogenous stem cells that rely on tissue digestion and adherence.10 How2 Stem cells from different parts of the body express different surface markers  markers, monocyte (CD14 or CD11b), B cell (CD79a or CD19) markers, and human leukocyte antigen (HLA) class II (HLA‐DR) surface antigens.13 Ste2.1 Ghazanfari et al. demonstrated that BMSCs expressing CD45−, CD271+, CD140alow/−, CD90+, and CD105+ have the potential for trilineage differentiation into adipose, bone, and cartilage tissue. CD271 is the most specific surface molecule used for the isolation of BMSCs. Compared with umbilical cord MSCs and ADSCs, BMSCs exhibit lower methylation chondrogenic production induced by the microenvironment and highly express COL2A, COL10A, ACAN, PTHR1, and DLX6, with a stronger chondrogenic ability. This indicates that the microenvironment surrounding BMSCs is closely associated with the expression of a specific endochondral differentiation pattern in the BMSCs. BMSCs are located in the subchondral bone, in marrow, and on the inner surface of the bone. They directly participate in the process of bone and cartilage remodeling without having to migrate to the periphery of vascular tissue. Adult chondrogenesis‐inducing injuries that can trigger the pathway of endochondral ossification cause BMSCs to migrate to the site of the injury to form a unique niche in the microenvironment, which in turn differentiate into an early cartilaginous tissue.BMSCs originate in the bone marrow cavity. Their stemness has been demonstrated by the formation of ectopic bone tissue from a single cultured BMSC.25 Gha2.2 Within the joint cavity, ADSCs originate from within the infrapatellar fat pad (IFP), where most stem cells are latent in the stromal vascular fraction (SVF) and they have some clinical application. Both ADSCs and BMSCs are multipotent; however, BMSCs are more committed to osteogenic and chondrogenic lineages, whereas ADSCs are more committed to the adipogenic lineage. ADSCs tend to differentiate into adipocytes and vascular endothelial cells and are affected by the SVF microenvironment. When exposed in vitro to transforming growth factor beta (TGF‐β), ascorbic acid, and dexamethasone, ADSCs expressing CD9+, CD10+, CD13+, CD29+, CD44+, CD49e+, CD59+, CD105+, CD36+, and CD166+ are more inclined to differentiate into chondrocytes. CD36+ and CD106− are specific markers for ADSCs. Compared with BMSCs, ADSCs are easier to obtain and exhibit higher proliferation ability. However, the application of ADSCs to tissue engineering depends more on the regulation of the microenvironment, which induces ADSCs to undergo programmed chondrogenic differentiation. For example, various factors, such as bone morphogenetic protein (BMP)−6, TGF‐β3, insulin‐like growth factor 1 (IGF‐1), and the presence of an alkaline environment substantially affect the collagen type I alpha 1 (COL1A1) and alpha 2 (COL2A1) contents of the cartilage tissue formed by ADSCs.ADSCs can differentiate into a wide range of cell lineages, such as bone, cartilage, vascular endothelium, liver, and nerve cells.32 Wit2.3 When cartilage is injured, more SMSCs are released from synovial tissue compared with adipose tissue, as SMSCs more readily enter the cartilage tissue. Of note, immediately separated synovial cells express CD10, CD13, CD14, CD34, CD44, CD45, CD49a, CD62e, CD73, and HLA‐DR surface markers. After passage, CD14, CD34, CD45, CD62e, and HLA‐DR disappear, CD105 and CD166 appear, and CD10, CD14, CD44, CD49a, and CD73 exhibit increased expression. SMSCs show little change from passage 1 to 8 and are characterized by the potential for chondrogenesis and differentiation. This shows the difference between resting and active SMSCs. When the microenvironment changes, SMSCs re‐adopt the characteristics of cartilage stem cells. In the specific matrix microenvironment of an extracellular matrix (ECM) occupied by SMSCs, adult SMSCs are restored to optimal vitality, thereby promoting cartilage regeneration.During the cartilage tissue repair process, SMSCs enter the superficial area to assist the resident cartilage progenitor cells to repair the cartilage defects.38 Whe2.4 SFSCs originate from an adjacent synovium or from a superficial layer of the cartilage. Whether in normal synovial fluid or in the synovial fluid of osteoarthritis (OA) patients, SFSCs express the following markers: D7‐FIB+, CD13+, CD105+, CD55+, low‐affinity nerve growth factor receptor (LNGFR)+, CD45−, BMP receptor type IA (BMPRIA)−, and CD10low/−. In the normal human knee joint, SFSCs account for 0.025% of the total cells in the joint. Compared with BMSCs, SFSCs have a tendency to form cartilage that may be related to higher hyaluronic acid (HA) content in the microenvironment around SFSCs. Because of the lack of a guiding microenvironment in the joint synovial fluid, stem cell fate cannot be guided. Compared with SMSCs, SFSCs are characterized by higher clonality and a more uncertain differentiation orientation.During the repair process, SFSCs, like SMSCs, are mainly responsible for repairing the superficial layer of articular cartilage.38 SFS2.5 In the resting state, cartilage stem cells, such as CSPCs, participate in the maintenance of homeostasis. CSPCs express many specific markers, including CD9, CD29, CD166, CD49e, and CD5. In the OA microenvironment, CSPCs also express Notch‐1, STRO‐1, and vascular cell adhesion protein 1 (VCAM‐1). CSPCs isolated based on these markers have the potential to differentiate into cartilage, fat, and bone, whereas their chondrogenic ability is higher than that of ADSCs. Downregulation of the Runt‐related transcription factor 2 gene enhances the chondrogenic differentiation potential of CSPCs. Following mechanical injury of the cartilage or in an OA environment, CSPCs are released from dormancy, participate in the early stage of cartilage repair, and upregulate the expression of ECM‐related proteins. Additionally, the 3D microenvironment, ECM components, TGF‐β3, and BMP‐6 can significantly influence the fate of CSPCs. In fact, the chondroprotective ability of CSPCs may be enhanced by inhibiting matrix degradation.Although articular cartilage lacks inherent repair ability, it has been proved that the superficial layer of articular cartilage contains CSPCs.48 In 2.6 SSCs express podoplanin (PDPN), CD146, CD73, and CD164 on their surface and are derived from fetal bone, adult bone, BMP‐2‐treated human adipose stroma, and skeletally differentiated induced pluripotent stem cells. Although SSCs may be enriched with the cell surface markers STRO‐1 and CD146+, individual cell‐specific markers that are expressed by SSCs remain undefined. SSCs can only differentiate into progenitor cells in the cartilage and matrix and do not produce adipocytes or endothelial cells. SSCs can also form bone and bone marrow matrix in nonskeletal tissues. The fate of SSCs may be guided by the microenvironment irrespective of whether it involves a change from bone to cartilage or vice versa.In recent years, researchers have further examined bone progenitor cells. For instance, Chan et al. identified SSCs.53 SSC3 The AIME primarily includes damaged cells, vasculature, infiltrating immune cells, danger (inflammatory) signals, and proinflammatory molecules ). The AIME plays three main roles: release of danger signals, recruitment and polarization of inflammatory cells, and removal of inflammatory factors. Early inflammation promotes the clearance of necrotic tissue, activation of immune cells, and recruitment of stem cells. During the middle and late stages, inflammation inhibits and disrupts the differentiation of stem cells. In terms of the differentiation and expression fate of the latter, stem cells first differentiate into fibroblasts, then into chondrocytes, and finally into osteoblasts. When the AIME is well regulated, inflammation rapidly resolves and the normal cartilage architecture is restored. When the AIME becomes imbalanced, rheumatoid arthritis, OA, hyperplastic osteophytes, or fibrotic scar tissues can develop, ultimately leading to the failure and death of the entire cartilaginous tissue. Therefore, maintaining homeostasis in the AIME as well as its proper intervention and control play crucial roles in cartilage repair and stem cell fate decisions. Temporal and spatial changes in the AIME are shown in Figure Because the joint cavity is a closed environment, inflammation triggered by most cartilage injuries is a form of aseptic inflammation.54 The3.1 DAMPs primarily use the nuclear factor kappa B (NF‐κB) and mitogen‐activated protein kinase pathways to regulate immune and some nonimmune cells. A decrease in S100 protein expression can significantly increase the activity of adipose stem cells. Conversely, an increase in S100 protein levels can induce stem cells to differentiate into osteoblasts and form ectopic osteophytes during the process of cartilage matrix remodeling. The excess accumulation of DAMPs leads to aging and a loss of stem cell activity, thereby leading to the adverse transformation of their cell fates, an imbalance of the AIME, and even the triggering of a systemic inflammatory reaction. Additionally, during the early stage of cartilage injury, the dynamic balance of ROS in the AIME is disrupted. With the increase in the number of dead cells, ROS such as hydrogen oxide (H2O2), superoxide anion (O2−), and hydroxyl radical (OH)− gradually accumulate in the AIME. Increased levels of reactive oxygen and nitrogen species (RONS) inhibit the proliferation and differentiation of MSCs by activating the NF‐κB pathway. The accumulation of RONS can also inhibit the survival of MSCs and the secretion of type II collagen.The excessive and chronic release of damage‐associated molecular patterns (DAMPs) can lead to abnormal inflammation of the joints and destruction of the cartilage. In the AIME, alarm proteins  are mainly derived from damaged or dead cells.61 DAM3.2 When the cartilage is injured, neutrophils, as an advance team, respond within 20 min and migrate within 2−6 h. Under the stimulation of inflammation, neutrophils release neutrophil extracellular traps (NETs) within 60 min. Neutrophils respond to DAMPs, accumulate at the injured site, release matrix metalloproteinase (MMP)−9, and clear cell debris. After the neutrophils mature, they are cleared by macrophages and home to the bone marrow. Mature neutrophils release liposomes to inhibit the entry of other neutrophils. By releasing TGF‐β, IL‐10, and other growth factors, they reprogram monocyte‐derived macrophages from the proinflammatory phenotype to the anti‐inflammatory phenotype. However, in cases of excess ROS accumulation or an unscheduled apoptosis of neutrophils, neutrophils release NETs to aggravate the tissue damage. The timely clearance of neutrophils and the low‐level secretion of NETs are the first step toward the formation of a beneficial AIME. This is critical for the activation and recruitment of stem cells.Following cartilage injury, the innate immune response cells are activated first, while macrophages, monocytes, and multinuclear neutrophils invade local tissues and release soluble inflammatory factors.69 Whe Natural killer (NK) cells can stimulate the recruitment of MSCs by releasing chemokine (C‐X‐C motif) ligand 7 (CXCL7), CXCL2, CXCL3, chemokine (C‐C motif) ligand 5 (CCL5), and IL‐8, thereby helping to activate stem cells and release interferon gamma (IFN‐γ) to resist further inflammation. Additionally, crosstalk between NK cells and MSCs affects the viability of MSCs to some extent.Mast cells, which are nonspecific immune cells, secrete MMPs, IL‐1, IL‐17, and other substances to recruit other immune cells when cartilage is injured, thereby promoting the degradation of dead chondrocytes and remodeling of the subchondral bone.77 Nat Macrophages in the articular cavity primarily come from the synovium, bone marrow, and IFP, including resident and recruited macrophages. The recruited major histocompatibility complex (MHC)‐II macrophages play a leading role in the regression of inflammation and reprogramming of the inflammatory phenotype. Within 72 h of cartilage injury, macrophages infiltrate the injured tissue and increase in numbers. Macrophages polarize to M1‐type macrophages  and secrete chemokines and MMPs to clear dead cells. During the middle and late stages of inflammation, macrophages polarize to M2 macrophages , secrete platelet‐derived growth factor and TGF‐β, and form an AIME that is conducive to the proliferation and differentiation of MSCs, while M2 macrophages promote the synthesis of ECM to facilitate wound contraction and healing. The timely transformation of the macrophage phenotype plays a decisive role in the formation of the AIME. M1 macrophage‐secreted tumor necrosis factor alpha (TNF‐α), IL‐1, and IL‐6 drive stem cells to differentiate into fibroblasts. However, studies have shown that TGF‐β, hypoxia‐inducible factor 1 alpha, IL‐4, and MMP12 are the main factors that drive macrophages to differentiate into (IL‐4) M subtypes and promote cell fibrosis. An increase in COL2A expression increases M2 macrophage differentiation, inhibiting apoptosis and the excessive hypertrophy of stem cells.Macrophages play a key role in maintaining the homeostasis of the tissue microenvironment in the cartilage.83 Mac Activated regulatory T (Treg) cells promote the expression of IL‐10 and TGF‐β and the secretion of indoleamine 2,3‐dioxygenase that inhibits the production of IL‐6, thereby maintaining the stability of the regenerative microenvironment and indirectly promoting tissue regeneration. In the AIME, CXCL10 binds to CXCR3 and cells that express CXCR3 (NK and activated T‐ and B‐cells) are attracted to the inflammatory site and indirectly affect the fate of stem cells. B‐cells secrete various proinflammatory factors  and induce chondrocyte death and cartilage matrix rupture. Mature DCs activate Th1‐ and Th17‐cells, thereby inhibiting the chondrogenic development of MSCs, resulting in cartilage degradation. Immature DCs not only promote Treg cell proliferation but also trigger MSCs to differentiate into chondrocytes through the secretion of IL‐10.In the AIME, T‐cells, B‐cells, and dendritic cells (DCs) mainly play the role of antigen presentation and chemokine secretion and indirectly regulate the joint microenvironment.87 Act3.3 The degradation proteins of the cartilage matrix, such as the MMP family, reach their highest levels at 24 days after the peak expression of inflammatory factors. The recruitment factor of macrophages (CCL2) reaches its peak within 72 h after injury. In the late stage of AIME imbalance, the accumulation and persistence of inflammatory factors further stimulates inflammatory cells to cause a cytokine storm, which not only inhibits the activity of stem cells but also affects the original cartilage tissue and causes extensive inflammation in the joints. Among these factors, IL‐1β, TNF‐α, IL‐6, and IL‐17 play a leading role in the AIME at the terminal stage. They can not only promote each other's release but also initiate the downstream inflammatory reaction. Both IL‐1β and TNF‐α inhibit the vitality of MSCs, reduce the production of glycosaminoglycan, increase the production of nitric oxide and MMPs, and further destroy the cartilage matrix. In fact, IL‐1β and TNF‐α and the accumulation of IL‐6 and IL‐17 amplify osteoclast activity and accelerate the necrosis and fibrosis of damaged tissues. Furthermore, IL‐18, MMPs, and Yes‐associated protein can inhibit the differentiation of stem cells into cartilage.Within 24 h after injury, the content of inflammatory factors  in the AIME increases significantly and peaks within 72 h. The content of these inflammatory factors can be higher than normal within 3 weeks of inflammation.96 The44.1Figure  DAMPs can activate multiple pattern recognition receptors (such as membrane‐bound Toll‐like receptors (TLRs) and C‐type lectin receptors, cytoplasmic NOD‐like receptors (NLRs), and retinoic acid‐inducible gene‐I‐like receptors) and some nonpattern recognition receptors . Therefore, these receptors can be targeted to inhibit inflammation progression and stem cell apoptosis. For example, A740003, MCC950, and A20 can regulate the AIME in OA models in vivo by inhibiting the NLR family pyrin domain containing three inflammatory bodies. The improved nucleic acid‐binding polymers can neutralize the activation of TLRs caused by DAMPs. Moreover, some cationic nanoparticles can bind cell‐free DNA (cfDNA) to block TLR activation. Liang et al. applied cationic nanoparticles to a rat model of rheumatoid arthritis and successfully relieved joint swelling and osteochondral injury. Additionally, small interfering RNA (siRNA) may also be designed to directly target the generation of DAMPs. Stimulator of interferon genes, cyclic GMP‐AMP synthase, and other small molecule inhibitors also eliminate the inflammatory reaction caused by DAMPs. Moreover, the generation of RONS can be eliminated through the use of active antioxidant materials. For example, nanoenzyme containing metal ions, such as Ce, Fe, Ce, Mn, V, Cu, and Co, can eliminate RONS through electron transfer with materials. Li et al. successfully alleviated joint hypoxia in a rheumatoid arthritis model using a nanoenzyme doped with concave cubic rhodium (Rh/SPX‐HAS), thereby improving the AIME. Kim et al. cleared ROS in a rat model of rheumatoid arthritis using manganese ferrite/cerium co‐modified nanoparticles, thereby alleviating the progression of arthritis.In the event of early danger signals in the AIME, the excessive release of DAMPs can be inhibited by blocking their receptors or action processes Figure 3a. The rFigure 54 DAM4.24.2.1 Bioactive factors, such as type I collagen, chondroitin sulfate, sulfated HA, and platelet‐rich plasma, can also be added to the material to indirectly induce the polarization of macrophages, promote the differentiation of stem cells, and protect the newly formed cartilage tissue from arthritis.In the AIME, macrophage reprogramming is a determinant of AIME condition Figure . The tra126 Bi4.2.2 Recently, MSC‐derived exosomes have become excellent tools to regulate the AIME. Extracellular vehicles (EVs) derived from ADSCs, BMSCs, or embryonic stem cells can inhibit the production and infiltration of M1 macrophages, increase the expression of cartilage ECM components, and promote the differentiation of stem cells into cartilage. Woo et al. successfully inhibited M1 macrophage infiltration in rat and mouse OA models treated with MSC‐derived exosomes, thereby ameliorating OA symptoms. Jiang et al. used Wharton's jelly MSC‐derived exosomes to successfully promote cartilage regeneration and improve the joint cavity microenvironment in rabbit and rat cartilage defect models. Interestingly, the type of stem cells used can only affect the production of exosomes but not the efficiency. Some adult cells also secrete exosomes. The exocrine activity of osteoclasts promotes chondrocyte hypertrophy. Exosomes derived from M1 macrophages aggravate the inflammatory reaction in the AIME; however, those derived from chondrocytes and M2 macrophages exhibited the potential to promote cartilage repair. Sang et al. successfully promoted the transformation of macrophages in the AIME toward the M2 type by carrying exosomes derived from primary chondrocytes using thermosensitive hydrogels, thereby alleviating OA. Da‐Wa et al. used M2 macrophage‐derived exosomes to inhibit the PI3K/AKT/mTOR pathway in the AIME, thereby inhibiting the inflammatory response and minimizing knee OA damage in a rat model.Exosomes derived from stem cells are AIME regulating substances produced via paracrine secretion and are an effective component of stem cell therapy. MSC‐derived exosomes can effectively reduce the production of inflammatory cytokines in chondrocytes, increase the expression of cartilage ECM components, and promote the regeneration of cartilage tissue.128 Re4.2.3co‐glycolide) or chitosan can accelerate the maturation of DC cells and increase the proliferation of T‐cells, release of inflammatory factors, and destruction of cartilage. HA and agarose can impede the maturation of DC cells, maintain the function and phenotype of DC cells at the level of immature DCs, and promote the differentiation of stem cells into chondrocytes.Macrophage polarization refers to the process wherein a macrophage displays different phenotypes (M1 and M2) corresponding to a different AIME. The transformation of phenotypes between M1 and M2 macrophages is important in the regulation of stem cell fate. Based on macrophage polarization, we can regulate other immune cells to achieve the goal of macrophage reprogramming. Poly as a carrier to deliver quercetin for the treatment of rat OA. Although these two materials are characterized by an excellent delivery performance, their hepatotoxicity remains a problem. As a highly programmable biocompatible material, structural DNA can achieve good targeting through the modification of aptamers. Ma et al. achieved a targeted antioxidant effect with DNA origami drugs. Engineering vesicles have the advantages of immune escape and low immunogenicity. Moreover, they can carry a wide range of molecules and effectively increase the delivered concentration. Li et al. used EVs to carry curcumin and to alleviate the progress of OA. Hydrogels, as a widespread and low toxicity molecular delivery material, have many advantages: they can be widely sourced, exhibit good biocompatibility, and are easy to synthesize. Yu et al. used hydrogel microspheres to repair cartilage in an OA model. Synthetic polymers can significantly prolong the retention time of drugs and determine the action time of drugs based on charge. Shen et al. used a synthetic polymer to wrap dexamethasone and achieve targeted and biological imaging of osteoarthritis. Plasma nanoparticles, such as gold nanoparticles (AuNPs), have obvious advantages for the delivery of negatively charged materials. Li et al. used AuNPs to develop an antioxidative and anti‐inflammatory treatment and reduced the toxicity of the AuNPs through surface modification.To enhance the function of small molecule drugs, the adoption of a design strategy to enhance efficacy is necessary Figure 4a. Therere Table 211 An4.5 Dynamic response materials can determine the response time point according to pH, redox reaction, enzyme, glucose, ions, adenosine triphosphate (ATP), anoxic environment, temperature, mechanical stress, and nucleic acids, which are parameters beyond the reach of conventional drugs  technology is gradually becoming the main choice for delivering precision medicine (Figure  Electrohydrodynamic jet 3D printing, low‐temperature deposition 3D printing, and microfluidic technology are effective means for achieving this goal. The biomaterials designed using LbL technology can adapt to the temporal and spatial changes of the AIME as well as to the different anatomical structures of the defect, thereby enabling the delivery of various bioactive molecules.With the maturity of AIME‐targeting technology, methods for regulating the AIME have improved. A dynamic regulation strategy of the inflammatory response based on nanomaterials can regulate the balance of the AIME.213 Dys Figure .[214e Figure .[2155Figure  Moreover, organ‐on‐a‐chip engineering technology based on microfluidics and 3D printing will enable further exploration of the influence of the AIME on specific cells (Figure Different AIMEs can play a decisive role in cell fate. Herein, we have discussed the influence of various factors on stem cell fate. An effective method to simulate this complex microenvironment is by constructing cartilage organoids and combining transcriptomics, proteomics, spatial transcriptomics, and machine learning methods to analyze and model the AIME Figure 5a.[219s Figure . More im One reason may be that the drug delivery strategy leads to the inability of inflammatory factors to target the desired site, thereby resulting in ineffective concentrations of inflammatory factors. Another reason may be that the inhibition of a single molecule is not sufficient to reverse the progress of the AIME imbalance during the late stage of inflammation. Delivering effective biomolecules with different enhancement strategies is an effective method for increasing drug concentration and targeting efficiency. However, evaluating the safety of small molecular particles and nanoparticles as well as the uncertainty of their therapeutic effects are factors that hinder their clinical transformation. Recently, bioactive molecules based on structured DNA and active peptides have exhibited great potential for use in cartilage repair. The deconstruction of small molecule peptides and development of methods for simulating nucleic acid molecular dynamics have provided a large research library for constructing new protein and nucleic acid molecules. We believe that the development of novel small molecule drugs will be significantly accelerated in the future.The use of a previous generation of biomaterials has generated significant knowledge. For example, in animal models, key factors of the AIME and novel therapeutic strategies have been verified. However, in clinical studies, the strategy of a single molecule‐targeted antagonism has not achieved satisfactory results Figure .[100,6The potential for cartilage regeneration is closely associated with the AIME. An ideal AIME following injury should progress through three stages Figure . The earThe authors declare no conflict of interest.


Maxillary molar distalization is a common technique used in the non-extraction treatment of Angle Class II malocclusion that can effectively correct the molar relationship and create spaces for anterior teeth alignment. However, this approach may also impact the temporomandibular joint (TMJ) due to predictable changes in the posterior vertical dimension. Despite its widespread use, Class II malocclusions correction by molar distalization with clear aligners has not been investigated for their effects on the TMJ. Therefore, this study aimed to analyze the impact of sequential molar distalization using clear aligners on the TMJ.Three-dimensional CBCT scans of 23 non-growing patients  with skeletal class I or II malocclusion and a bilateral molar class II relationship treated by sequential upper molars distalization with orthodontic clear aligners . A total of 46 joints were examined before and after molar distalization using Anatomage InvivoDental 6.0.3. Linear and angular measurements of the mandibular joint were measured, including joint parameters, inclination, position, and the dimension of the condyle and articular fossa. In addition, 3D volumetric spaces of the joint were analyzed. All data were statistically analyzed by paired T test to determine the differences between the pre-and post-orthodontic procedures.P ≤ 0.05.No statistically significant differences were found in all primary effects resulting from maxillary molars distalization by clear aligners on TMJ components measurements and joint spaces between T0 and T1. Meanwhile, statistically significant differences were observed in the linear position of the upper molars and the molar relationship parameter with at least Treatment by sequential upper molars distalization with clear aligners does not lead to significant TMJ parameters changes in condyle and fossa spaces, dimensions, and positions.The online version contains supplementary material available at 10.1186/s40510-023-00474-3. Malocclusions have become a growing concern in oral public health. According to the World Health Organization, malocclusions are now recognized as the third most prevalent oral health concern, following dental caries and periodontal disorders . In partSince extractions treatment has been related to adverse side effects such as facial profile worsening and TMJ problems , distaliHeadgear was the first appliance used for molar distalization and has been the most frequently used appliance to correct anteroposterior discrepancies since the 1950s. However, this appliance requires substantial patient cooperation and is esthetically undesirable . In receClear aligners are orthodontic treatment systems introduced as more aesthetically pleasing and convenient substitutes to conventional fixed appliances. They can address various types of malocclusions, including treating class II malocclusion in adult patients through sequential maxillary molar distalization , 13.The association between dental occlusion and temporomandibular disorders (TMD) remains a controversial issue in dentistry. Thus, Manfredini et al.  conducteBackward positioning of maxillary arch molars can result in an alteration to the position of the teeth and inter-arch relationship, which can cause repositioning of the mandible and potentially affect the position of the condyle. This, in turn, may disrupt the disc-condylar relationship and induce TMD. In addition, patients undergoing orthodontic treatment to correct malocclusions often experience TMJ adaptive bone remodeling , 21. TheVarious methods have been used in orthodontic research to visualize changes in the treatment of temporomandibular joint resulting from functional treatment, such as cephalograms , 27, panCBCT scans provide accurate and precise quantitative data, allowing for comparisons of images without magnification and making them a valuable tool for analyzing treatment outcomes. These scans can also assist in volumetric measurements and can evaluate changes in the contours and forms of objects, which are often limited in 2D cephalometry. Moreover, CBCT scans provide more data than 2D images , 35. In Based on the authors’ knowledge, this is the first study to evaluate the TMJ structure changes three-dimensionally following sequential molar distalization of the upper arch using clear aligners to correct class II malocclusion. Thus, this study aimed to three-dimensionally analyze the impact of sequential molar distalization using clear aligners on TMJ.This retrospective study analyzed CBCT images of a sample of 23 non-growing subjects  treated with sequential molar distalization using orthodontic aligners . All procedures were conducted according to the Helsinki Declaration, and written consent forms were signed by all patients. Ethical approval was granted by the ethical committee of Lanzhou University’s School of Stomatology, Lanzhou, Gansu Province, China . The sequential upper molars distalization treatment. Figure Inclusion criteria for all subjects were as follows: (1) over the age of 18, (2) with skeletal class I or class II malocclusion and a bilateral molar class II relationship, (3) all permanent teeth, except the third molar, have erupted, (4) no history of TMD symptoms in accordance with TMD Diagnostic Criteria , (5) gooThe exclusion criteria were as follows: (1) under the age of 18, (2) imaging manifestations of condylar degenerative conditions , (3) extraction treatment except for third molars, (4) functional mandibular deviations or facial asymmetry, (5) surgical history at craniofacial region or TMJ, (6) any systemic disease or chronic medication use, (7) and skeletal malformation in the craniofacial region. Gender differences were not examined since only non-growing patients were involved in this study.α = 0.05) and effect size (dz = 0.8), where the mean values of (MR) were 3.1 ± 1.4 and 1.2 ± 0.6 for pre- and post-treatment, respectively. The analysis indicated that at least 19 subjects are required. The sample size included in our study was 23 subjects.The sample size of the present study was estimated based on the study of  using the G*Power 3.0.10. software  depending on the molar relation (the primary outcome of this study). The a priori sample size calculation was performed with a power level of 95% at a 5% significance level  was used to execute CBCT. All patients were scanned with standard protocol: field of view (FOV) was 16.0 × 13.0 cm, the setting of exposure parameter was 18.54 MAs; 8.9 s; 120 kV, and image acquisition at 0.4 mm voxel size. Moreover, with head position standardization, Frankfort horizontal plane (FHP) parallel to the floor, and maximum occlusal intercuspation. According to the imaging protocol, the patients were asked to prevent from swallowing or moving throughout the scanning process.Digital Imaging and Communications in Medicine (DICOM) files of the CBCT images were obtained and then introduced into version 6.0.3 of the InVivoDental, (Anatomage Inc.) software program for the linear and angular three-dimensional and volumetric analysis.The applied standard and innovative 3D TMJ analysis method was adopted from Alhammadi et al. –42 to meThe 3D skeletal, dental, and TMJ landmarks are shown in Tables The 3D analysis was designed based on the determination points in this sequence. First, the coordinate system’s orientation is set according to facial skeletal points of midline: nasion, basion, and incisive foramen, which were proved as valid points by Green et al. , and theA prior study by Abdulqader et al.  was publFor method error verification, the intra- and inter-examiner reliability of the measurements was analyzed by retracing 10 cases by two different observers within 2 weeks. Intra-class correlation coefficients (ICCs) and the absolute and relative technical error of measurement (TEM and rTEM) were calculated to determine the reliability and reproducibility of the measurements. Bland–Altman plot was also used to assess the reproducibility and reliability of TMJ landmarks  was used for statistical analysis. GraphPad Prism 8 was used to plot the graphs. Data were checked for normal distribution using the Shapiro–Wilk test. For each variable of 46 joints, descriptive statistics of the mean and standard deviation (SD) were calculated. The paired T test was employed to examine the differences between the two sides’ TMJ parameters before (T0) and after (T1) molar distalization periods. The significance level was chosen at Descriptive data among the 23 adult patients with class II malocclusions fulfilled the inclusion and exclusion criteria. The frequency of molar relationship Class II regarding its severity was as follows: six subjects 1/4 cusp, nine subjects 1/2 cusp (end-to-end), and eight subjects full cusp Class II relationship. In addition, 7 and 16 subjects were males and females, respectively, aged 22–47 years, with an average age of 29.8 ± 4.6 years. The means and standard deviation for each variable of recorded data are presented in Table P > 0.05), as shown in Fig. P ≤ 0.03) by 2.54 and 2.18 mm, respectively, as shown in Fig. No statistically significant differences were found in the net impacts resulting from maxillary molars distalization by clear aligners on the osseous mandibular joint’s components and joint spaces on both left and right sides of patients before and after treatment  dimensions, no statistically significant differences were found between T0 and T1. Moreover, no statistically significant changes were found for the mandibular condyle inclination, position, and dimension between before and after maxillary molar distalization of the treated sample. Similarly, no statistically significant variations were observed for the analyzed volumetric total joint space and other TMJ space measures.Intra- and inter-observer reliabilities analysis data of all the TMJ measurements are presented in Table The TMJ pretreatment values could be used to assess TMJ changes and evaluate treatment outcomes after orthodontic or orthognathic treatment in young adults. The detailed measurements of the TMJ’s anatomical structures in three-dimensional planes of their interpretations will help understand TMJ’s pathological alterations .The excellent correlation coefficient between intra- and inter-observer reliability measurements indicated high and precise landmark identification with CBCT, which is regarded as an ideal tool for osseous assessment of the anatomic structures and cannot be obtained with any other conventional modality used to evaluate the complex temporomandibular region , 46.As the first study that three-dimensionally evaluated the TMJ structure changes using CBCT before and after sequential molar distalization of the upper arch by clear aligners for correcting class II malocclusion, the result of this study will be helpful in clinical treatment planning for asymptomatic patients or patients with subjective TMD symptoms who proposed to undergo orthodontic treatment.In an adult, class II correction mainly results from tooth movement without the effects of growth, and molar distalization is often undertaken to gain 2 to 3 mm of space in the dental arch in order to obtain a class I relationship . In clasResults indicated the potential of maxillary molar bodily movement, at least when a minimal sagittal plane correction is needed, whereas our sample included subjects with multiple complexities of Class II molar relations varied from 1/4 cusp to full cusp of Class II molar relationship. A significant distal movement was observed of the upper molars and the related correction in the molar relationship (MR) with the absence of changes in mandibular fossa dimension and condylar inclination, position, and dimension outcomes for pre- and post-molar distal movement, thereby confirming the capability of performing a distal body movement of the upper molars by clear aligners with complete control of the TMJ measures the opposite of what has been reported with other orthodontic appliances . The posFurthermore, no significant change has been demonstrated regarding the mandibular fossa dimension. Similarly, no significant differences were observed between the pre-and post-treatment groups in condylar inclination and condylar dimension for both sides after correcting class II malocclusion by Invisalign aligners.Anterior or posterior condyle position may directly affect facial morphology , 51. In Lione et al. reported that clear aligners provide better vertical dimension control during distal teeth movement. The thickness of aligners and the impact of the biting block of aligner material may explain the nonexistence of a significant vertical dimension increase . The insAs for TMJ spaces, no statistically significant differences were observed in both sides of anterior, posterior, superior, and medial joint spaces before and after the treatment by distalization of upper molar teeth with clear aligners. However, volumetric joint space effects of molar class II correction with aligners have never been described . The preLooking at the results of this study, the upper molars distalization technique performed with clear aligners seems to overcome various side effects related to this orthodontic procedure typically observed with other appliances , 49 and Accordingly, orthodontic aligners could represent an effective option for molar distalization approach, especially for TMJ pathologies subjects, at least for molar distal movements up to 2–3 mm.This work is limited by its low sample size. In future studies, the mean amount of distal movement should be increased with various groups of malocclusion comparison to validate the control of TMJ structures after molar distalization using clear aligners.The study revealed insignificant changes in condyle-fossa spaces, dimension, and position in patients treated for class II malocclusion with sequential molar distalization using clear aligners, indicating that clear aligners do not significantly impact TMJ parameters during or after sequential molar distalization. Accordingly, sequential molar distalization using clear aligners is a viable treatment option for class II malocclusion patients without adversely affecting TMJ parameters. However, orthodontists need to consider the effects of various orthodontics appliances on TMJ components when prescribing treatment for their patients.Additional file 1. Appendix A: Supplementary data.