Spatiotemporal patterns and trends of COVID-19 at a local spatial scale using Bayesian approaches are hardly observed in literature. Also, studies rarely use satellite-derived long time-series data on the environment to predict COVID-19 risk at a spatial scale. In this study, we modelled the COVID-19 pandemic risk using a Bayesian hierarchical spatiotemporal model that incorporates satellite-derived remote sensing data on land surface temperature (LST) from January 2020 to October 2021 (89 weeks) and several socioeconomic covariates of the 140 neighbourhoods in Toronto. The spatial patterns of risk were heterogeneous in space with multiple high-risk neighbourhoods in Western and Southern Toronto. Higher risk was observed during Spring 2021. The spatiotemporal risk patterns identified 60% of neighbourhoods had a stable, 37% had an increasing, and 2% had a decreasing trend over the study period. LST was positively, and higher education was negatively associated with the COVID-19 incidence. We believe the use of Bayesian spatial modelling and the remote sensing technologies in this study provided a strong versatility and strengthened our analysis in identifying the spatial risk of COVID-19. The findings would help in prevention planning, and the framework of this study may be replicated in other highly transmissible infectious diseases. It is highly transmissible by either direct contact with an infected individual or transmission via contaminated surfaces leaving the world in the last two years at a halt in many aspects5. In Canada, over 3.4 million COVID-19 cases and over 37,485 COVID-19 related deaths have been reported, with Ontario and Quebec reporting the highest cumulative cases in the nation6. Over 11.2 billion COVID-19 vaccine doses have been administered around the world7, and at least 89% population over the age of 5 years in Canada have received at least one dose 8. In Canada, Toronto continues to experience substantial COVID-19 incidence and hospitalization rates despite several interventions and mitigation efforts made by the local and provincial public health officials10.COVID-19, caused by the coronavirus SARS-CoV-2, has complex transmission dynamics possibly generated by different risk factors such as demographic, social and environmental factors11. A county-level study in the USA found a strong negative influence of nighttime land surface temperature (LST) with COVID-19 using low-resolution Moderate Resolution Imaging Spectroradiometer (MODIS) images of 202012. Another study found LST to be an important determining factor in the COVID-19 infection rate in Kolkata, India13. Another study by Hassan et al. identified a strong positive relationship between COVID-19 and LST14. This study showed that a 1 °C increase in LST is linked with a 36.1% increase in COVID-19 incidence rates in Bangladesh. Many prior studies have discussed the associations between temperature and COVID-1915; however, most of these studies have used short temporal periods, lower resolution images or have not performed a small area analysis. The results were diverse and often contradictory in different geographic areas, leaving a gap in understanding the impact of temperature on COVID-19 transmission in a small urban area.As momentum grows to end this global pandemic, understanding the disease trends, detecting hotspots, and identifying important risk factors at the community level is an imperative research effort. Temperature is often a significant risk factor for infectious diseases because a certain temperature can help a virus evolve rapidly20, rate of education22, ethnicity status28, immigration status33, unemployment rate34, and housing conditions30, tend to experience higher rates of COVID-19 infections and morbidity due to the economic and health inequalities. A previous work by Vaz in 202135 found that the COVID-19 cases are not uniformly distributed in Toronto. Social injustice, socioeconomic vulnerability and population density were found to be related to the increasing spread and incidence of COVID-19. A work by Feng in 2021 has found that neighbourhood-level population density and low income have a significant effect on COVID-19 mortality risk36. Another study of Toronto neighbourhoods by Choi et al. in 2021 found that several demographic and socioeconomic factors such as higher education rate, lower rates of immigrants (foreign-born residents) were significantly associated with decreasing the number of COVID-19 infections37. These past studies implied that these factors might disproportionately impact COVID-19 infection rates. Finally, analyzing the spatiotemporal trends of COVID-19 transmission to understand whether the disease risk trends show increasing, decreasing or stable patterns over the study period has also been understudied.Previous studies have also linked different socioeconomic and demographic factors to explain the heterogeneity in COVID-19 rates across space. Some studies have found that areas with low socioeconomic status, such as rate of poverty38 since it has the capacity to account for missing data, measurement errors and ecological bias40. Even though Bayesian models have a clear advantage, only a handful of studies45 in COVID-19 research has adopted Bayesian approaches to predict COVID-19 risk, identify trends and locate hotspots. Motivated by the recent increase in COVID-19 incidence in winter, this study scrutinizes the effect of weather and socioeconomic and demographic factors on COVID-19 using a small area analysis. Within the scope of this study, we will answer four research questions: (1) where were the hotspots of COVID-19, (2) what was the temporal patterns of risk in the study area, (3) was there a relationship between land surface temperature and COVID-19 while adjusting for socioeconomic and demographic factors, and (4) was there any spatiotemporal trend of COVID-19 transmission in Toronto ?In this study, we used a Bayesian hierarchical spatiotemporal models to investigate the spatiotemporal patterns of COVID-19 transmission in Toronto. The approach allows us to deal with uncertainties related to the data, the process and model parameters2 and a high population density of 4692 persons/km21. Toronto has a well-defined urban heat island with warmer temperatures, mostly at night and in winter, compared to the rest of the city’s surrounding regions4. The average temperature of Toronto is 21.9 °C (81.3 °F), and the annual rainfall is 845 mm (33.3 in.), with July (17–25 °C/62 °F to 77 °F) the hottest and February (average − 4.4 °C/24.1 °F) the coldest months of the year5. The map of the study area was created using ArcGIS Desktop software46 version 10.8.1 is provided in Fig. The study area is the metropolitan city of Toronto, located on the northwestern shore of Lake Ontario at an altitude of 175 m (43° 42′ 00″ N latitude and 79° 24′ 58″ W longitude). It is the capital city of the province of Ontario in Canada, with a total land area of 630 km47. We used the COVID-19 case data originally collected by Toronto Public Health and extracted from the provincial Case & Contact Management System (CCM)6 by the city of Toronto. The case dataset contains the demographic, geographic, and severity information for all confirmed and probable, sporadic, and outbreak-associated cases. We aggregated the daily case data from January 2020 to October 2, 2020, by the 89 epidemiological weeks and at the neighbourhood level. The total population for each neighbourhood was collected from neighbourhood profiles using the census 2016 population dataset collected and released by Statistics Canada48.There were 140 geographically distinct neighbourhoods in the study area. The digital data of the geographic boundaries for these neighbourhoods were acquired from the open data portal of the city of Toronto29. Based on these past studies and data availability, we selected 11 potential demographic and socioeconomic variables under six categories  for our model. These data were collected from the Toronto neighbourhood profile based on the 2016 census from Statistics Canada8. The full descriptions of the variables are provided in Table Previous studies have also linked different socioeconomic and demographic factors to explain the heterogeneity in COVID-19 rates across spaceWe used Landsat imageries to retrieve the weekly average land surface temperature (LST) at the neighbourhood level for 89 consecutive weeks . The timing of these imageries was approximately 4 PM (GMT). We collected mostly Landsat 8 imageries (75), and if the imageries for a particular week were not available in the Landsat 8, we used Landsat 7 imageries instead. However, no images were available for two epidemiological weeks (week 45 of 2000 and week 7 of 2021). We used the average temperature of the previous and following week for these two weeks. The images were corrected using atmospheric correction parameters collected by the National Aeronautics and Space Administration (NASA) to improve estimation accuracy. The complete details of the LST retrieval process, image data, acquisition time, atmospheric parameters are summarised in Table 46 version 10.8.1 to calculate the average LST values for Retrieval of spectral radiance. We applied a mask comprised of the polygons (neighbourhood boundaries) from the map of the city of Toronto and used the zonal statistics to calculate the average LST of all pixels by neighbourhood.Zonal statistics was performed in ArcGIS Desktop software59. We observed that the percentage of immigrants had a high correlation (> 0.7) with the percentage of the black population and the percentage of visible minorities. Since Choi et al.37, in a study conducted in our study area in Toronto, stated that the percentage of immigrants is an important risk factor in our study area in Toronto, we selected this variable over the percentage of the black population and percentage of visible minorities. The prevalence of low income was found to be strongly correlated with the rate of unaffordable housing and unemployment rate. Out of the three core housing need variables, the rate of unsuitable and the rate of unaffordable housing had a strong correlation with multiple variables. Since the rate of inadequate housing did not have a strong correlation with any other variables, we selected this (inadequate housing) variable over other housing variables. Among the correlated factors, we selected the prevalence of low income over the unemployment rate based on an earlier study in our study area that found low income to be strongly associated with COVID-1936. Finally since the LST and population density did not have a strong correlation with other factors, we kept both in the model.Variable selection was conducted using a two-step method to fit into the multivariable regression model. In the first step, we performed a Pearson’s correlation and generated a correlation matrix taking account of all the potential risk factors to evaluate collinearity among these risk factors Appendix . Note th28 in RStudio60 version 2021.09.0 to select the variables that fit best in our Bayesian hierarchical spatial model , the prevalence of low income, rate of higher education, inadequate housing, percentage of immigrants and population density per square kilometres, were fitted in a Bayesian variable selection method using BayesVarSel packageAppendix , suggestSince the variables were in different units, such as raw values, percentages, and prevalence rates, we used the Z-transformation technique, where the mean for all values was subtracted from each value and was then divided by the standard deviation of the values of the variables to obtain standardized values for the Bayesian model.38 and the space–time inseparable models63. Model 1 drew the space–time separable framework, while Models 2, 3 and 4 drew the space–time inseparable modelling frameworks.We performed four Bayesian hierarchical space–time models to investigate the long-term spatiotemporal effects of COVID-19 using two frameworks: space–time separablet in the neighbourhood i , the data model takes Poisson distributions as the likelihood in Eq. , where the variability of data is not captured by the space–time separable structure. Models 2,3, and 4 capture the space–time inseparable structure proposed by Knorr-held63, using three different space–time interaction effects that allow space–time inseparability. Table 31.Specifically, Poisson mean, entclass1pt{minimaentclass1pt{minima35 (Appendix The space–time separable and the three space–time inseparable models were fitted with Markov Chain Monte Carlo (MCMC) with different initial values for each model with a burn-in period of 116,000 iterations and thinning rate of 10 to obtain the posterior distributions of model parameters using WinBUGS software version 1.4. Mixings were observed using trace plots and autocorrelation plots. Convergence was evaluated by checking the Gelman–Rubin statisticAppendix . After tDIC) and the probability of direction (pD) values from the outputs to evaluate the goodness of fit for the four Bayesian hierarchical models and to select the best-fitted model64.We assessed the Deviance information criteria  outputs. Spatial relative risk  pattern of the relative risk for COVID-19 derived from the Bayesian model. The joinpoint regression uses an algorithm that tests whether a multi-segmented line is a significantly better fit than a straight or less-segmented line. It involves fitting a series of joined straight lines on a log scale to the trends in the weekly relative risk of COVID-19. Line segments are joined at points called joinpoints. Each joinpoint denotes a statistically significant (P = 0.05) change in trend. The significance test uses a Monte Carlo Permutation method to find the best fit line for each segment. The temporal patterns of the relative risk was plotted using the JoinPoint software.We used the joinpoint regression using the Joinpoint software version 4.9.0.00 and mapped in ArcGIS Desktop software46 version 10.8.1. The neighbourhoods with a negative estimated coefficient were considered to have a decreasing trend over time, and the neighbourhoods with positive estimated coefficients were considered to have an increasing trend over time. The neighbourhoods with zero estimated coefficients were considered to have a stable trend over the study period.The spatiotemporal trend of the relative risk over time  in the neighbourhoods was calculated in RStudio Software31, two additional models with two alternate prior assumptions than the original final model (Model 3) were run to perform a sensitivity analysis to examine the final model results. The first model was run with hyperprior distributions of Gamma 68 on the precision parameters  cases due to missing neighbourhood information, leaving 154,323 sporadic cases for the analysis.We had 2,731,571 population in the study area based on the 2016 census data. A total of 179,072  cases were reported in the 140 neighbourhoods during the study period. To avoid potential bias towards finding the high-risk clusters in the outbreak areas, our study excluded the outbreak-associated cases , generally from healthcare  or congregated settingsWe observed a sharp increase in COVID-19 cases in late December of 2000, which declined in early January 2021 Fig. a. Again,DIC values from the space–time inseparable models (Model 2–4) are much lower than the space–time separable model (Model 1), suggesting that the inseparable models  are better supported by the observed data, confirming the need to incorporate the space–time interaction component38. Finally, by comparing Model 2, 3 and Model 4, we found that Model 3 has the smallest pD, and DIC values, suggesting that Model 3 (space–time inseparable model with type II interaction) is the most parsimonious, and therefore was selected as our final model.We fitted the data in four Bayesian Space–Time Hierarchical Models and compared the models Table . The DICBetween January 21, 2020, and October 2, 2021, using the parameter estimates  was observed between March 14, 2021, and April 17, 2021. A total of 46 (51%) weeks out of the total 89 weeks experienced a relative risk of less than one during our study period.Figure Figure The posterior means of the relative risks  of COVID-19 in the Toronto neighbourhoods are presented in Fig. 65 we observed that only three neighbourhoods  had a decreasing trend of the relative risk during the study period 38 priors for standard deviation parameters are equally appropriate, giving almost identical posterior distributions of the parameters and model fit  compared to outputs of the original model (Model 3), presented in Appendix The sensitivity test models with Gamma We mapped the spatial patterns of COVID-19 cases during the post-study period  to visually compare the spatial patterns of risk identified from the Bayesian spatiotemporal model  of 2020 and 2021, which could be due to an increased time spent in outdoor settings leading to a decreased COVID-19 risk70. We identified significant changes in the risk of COVID-19 six times across the study period with varying trends. These changes suggest that the temporal trend of the epidemic of COVID-19 is different from other coronavirus diseases, such as the SARS (severe acute respiratory syndrome) epidemic in 2003, where the number of reported SARS cases has increased exponentially over time, and the outbreak lasted approximately 6 months74.The temporal risk was particularly high in the early spring of 2021, suggesting that the temperature during the season could have influenced the transmission of the disease in that part of the country. However, some other factors, such as an increase in mobility or travel patterns during the long weekend in March 20219. One reason behind this could be that high LST favours the coronavirus. Furthermore, Toronto is the most densely populated urban city in Canada. A recent study conducted in Phoenix, Arizona and Dallas, Texas, in the United States by Moss and Kar75 concluded that the urban areas that are susceptible to a high Urban Heat Index, as measured by LST, are primarily occupied by vulnerable population groups. Therefore, it is possible that an excess vulnerable population in an area creates a higher LST in the neighbourhood, resulting in an increase in the risk of COVID-19 in our study area.The findings of the positive association of COVID-19 with LST in our study are consistent with the findings of an earlier study22. It is likely that people with higher education may understand and follow public health messages as well as have the option to work remotely and maintain social distancing, resulting in lower incidences of COVID-19 in areas with a higher number of educated people. Additionally, a study by Mondal et al. in 2021 found that higher education levels were associated with a higher likelihood of vaccine acceptance76, suggesting that intervention with health education may play a key role in fighting this pandemic. Various public health programs such as COVID-19 awareness and health education programs in neighbourhoods with low education may also help reduce the fast transmission rates in those neighbourhoods.Our study also finds that the higher education rate has a negative association with COVID-19, which is in line with the findings from earlier studies77, which provided a high estimation accuracy. Our findings may advocate for maintaining disease surveillance and planning for an effective public health program. Most of the earlier studies82 explored the relationship between ambient temperature or LST and COVID-19 at a broader spatial scale  and used a shorter period with a very limited number of images to extract LST without any atmospheric corrections13. We believe that our study filled the gap in the existing literature by using higher spatial and temporal resolution satellite imageries at a local spatial scale, which is more spatially representative and may have provided a more accurate estimate due to the use of the atmospherically corrected data on LST.In our study, we used higher spatial and temporal resolution satellite images to extract LST. We also used atmospheric corrections methods on these images by adopting Sobrino et al. in 2008’s Land Surface Emissivity (LSE) modelThe validation results of the spatiotemporal patterns of risk using the data of the post-study period suggest that the Bayesian model could predict spatial patterns of risk for COVID-19 in our study area. Therefore, the findings of our study can be useful for increasing awareness of the disease and preparing public health interventions aimed at targeted prevention and control of COVID-19. Given limited resources available, efforts could focus on the high-risk neighbourhoods, as observed in our study.83 or lacks accurate information on the onset of the COVID-19, limiting the capacity of the analysis. However, the Bayesian spatiotemporal hierarchical models allowed us to compensate for the missing/unobserved covariates or missing data by incorporating the structured, unstructured random effects into the model63. In particular, the Type II space–time interaction in our final model implied that the missing covariates have smoothly varying structures through time and have no structure over space since they are highly localized in their effect on the outcome63. Third, our study has an ecological study design where the data were aggregated at the neighbourhood level, which may create issues such as ecological fallacy84. Therefore, these results cannot be interpreted at the individual level.Our study has several limitations. First, we could not find data on air pollution or human mobility patterns at the neighbourhood level, which could be important contributors to influencing the COVID-19 incidence. Second, we had 1.6% of cases with missing neighbourhood information and 12.4% cases were outbreak cases, which were not included in our analysis. Additionally, COVID-19 is often asymptomatic, under-reportedDespite these limitations, our study, due to its strong versatility and complex hierarchical modelling, is still convincing and has provided important information that may improve our understanding of the transmission patterns of COVID-19 and the associated risk factors. Also, our model is superior to the frequentist method that is more frequently used, as the Bayesian approach allowed us to compensate for the missing covariates in the models in identifying spatial patterns of risk. Therefore, we believe that using the Bayesian spatiotemporal model and the long-time series satellite-derived environmental data for modelling disease transmission have advanced our understanding of the disease risk in space.Several conclusions can emerge from our study. First, the Bayesian analysis has shown that Bayesian regression with spatial (structured and unstructured), temporal and spatiotemporal random effects provided an effective framework for understanding COVID-19 disease transmission. Second, the spatiotemporal risk remained high for the entire study period and constantly high for the high-risk neighbourhoods. However, the temporal risk fluctuated over time in the study area. Third, higher education and LST played an important role in predicting COVID-19 incidence. Therefore, it is important to take those factors into account while planning intervention strategies. Fourth, the framework presented in this study may help make an early warning system for COVID-19 incidence and assist public health authorities in controlling and preventing outbreaks of similar diseases. Finally, the methodological framework applied here can also be used in other small area-level studies on infectious diseases.Supplementary Information 1.Supplementary Information 2.


The tumor microenvironment (TME) acts as a crucial role in the occurrence and development of osteosarcoma (OS). Despite this, the mechanism controlling the components of immunity and stroma in the tumor microenvironment remains a mystery. To conduct this study, we download and collate transcriptome data from the TARGET database, whose full name is Therapeutically Applicable Research to Generate Effective Treatments, as well as available clinical information of OS. The CIBERSORT and ESTIMATE methodology are used to acquire the proportions of components of immunity and stroma and tumor-infiltrating immune cells (TICs). Protein-protein interaction (PPI) networks and Cox regression analysis are used to select differentially expressed genes (DEGs). A prognostic biomarker is determined by intersecting univariate COX and PPI results, which lead to the finding of Triggering receptor expressed on myeloid cells-2 (TREM2). Based on the next analysis, TREM2 expression is positively correlated with OS survival time. Immune function-related genes have enrichment in the group with high expression of TREM2, according to gene set enrichment analysis (GSEA). The percentage of TICs by CIBERSORT methodology revealed that the expression of TREM2 is positively associated with follicular helper T cells, CD8-positive T cells, and M2 macrophages and negatively correlated with plasma cells, M0 macrophages, and naive CD4-positive T cells. All results suggest a possible integral role of TREM2 in the immune-related events of TME. Therefore, TREM2 may be a potential indicator of remodeling of TME in osteosarcoma, which is useful and helpful in predicting the clinical prognostic outcome of OS patients and provide a unique perspective for immunotherapy for OS. Chiefly affecting children and young adults and occupying about nine percent of cancer-related deaths in youngsters whose age range is between 10 and 24 years old . With thActing decisive roles in tumor occurrence, progression, metastasis, and sensitivity to therapy, TME has aroused tremendous interest in basic and clinical research as a therapeutic target in cancer . ResidenIn our study, CIBERSORT and ESTIMATE methodology is used to count on the proportions and composition of the components of immunity and stroma of OS patients from the TARGET database and selected interesting biomarker TREM2. Several researchers identified the TREM2 receptor as a dominating signaling hub of pathology-induced immunity, which can sense tissue damage and activate robust remodeling immunity as responding to it . By playhttps://ocg.cancer.gov/programs/target) on May 24, 2022. In the genetic screening phase, we used all transcriptomic data, but in the prognostic analysis, we used only those data that had both transcriptomic data and survival status . After integration, a total of 95 samples had both transcriptome and survival data. There were 55 men and 40 women. Eleven patients were younger than 10 years, 62 were between 10 and 18 years, and 22 were older than 18 years.All data of transcriptome RNA-seq of 101 OS samples and clinical data (including age and sex) of 253 clinical cases are downloaded and collated from the TARGET database  are regarded as significantly differentially expressed genes.The median value allows the sample to be divided into two equal parts, so we use it as the split line. One hundred and one patients are distinguished as low or high scores, respectively, in comparison with the median ImmuneScore and StromalScore values. Differences between high- and low-scoring samples are achieved by using the R and limma package, also low and high subgroups are compared to obtain the corresponding differentially expressed genes. Genes with FDR <0.05 and logp and q values < 0.05 are regarded as an important and statistically significant role in the development and progression of osteosarcoma.The enrichplot, clusterProfiler, and ggplot2 packages of R software are used to classify 118 DEGs according to genomic annotation information, i.e., gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). Genomic annotation information with both R with the heatmap package is applied to establish the heatmap of DEGs.Data on clinical information of OS patients are also of interest. R software is used to perform statistical analysis, and Wilcoxon or Kruskal–Wallis rank sum tests are used to determine whether there are statistical differences between clinical indicators between two groups.PPI networks reveal the interactions between proteins, and we chose to use the STRING database to construct the corresponding network graphs. What is worth mentioning is that the nodes used to set up the network contain only those nodes whose confidence level of interaction is greater than 0.9.p  <  0.05 in both analyses of univariate Cox and Kaplan–Meier tests.Univariate Cox regression analysis allows initial screening out of potentially nonsignificant variables, which is achieved through R software and survival package. As shown, those ascertained and significant genes met p  <  0.05 and FDR q < 0.05 are regarded as significant sets. All GSEA analyses are performed on GSEA-4.2.3 software.Briefly, GSEA can determine the contribution of a predefined gene set to the phenotype, our gene set is all transcriptomic data as described previously, and the analysis is based on the C7 and HALLMARK target sets (v6.2). Just gene sets with corrected p < 0.05 are retained for subsequent analysis.The TIC abundance profiles of tumor samples can reflect the immune cell composition in osteosarcoma to some extent and can be calculated by CIBERSORT. The calculated results are screened, and only samples of p value <0.05 was considered statistically significant.All statistical analyses were conducted by R software (version 4.1.3). The Wilcoxon test was used to compare the differences between the two groups. This study can be divided into two major steps: the discovery of TREM2 and the follow-up study of TREM2 . First, p value is still less than 0.1. These entire results implied that the composition of TME is clinically important and the compositional aspects of TME can forecast patients' prognosis of OS, especially immune and stromal components.An important indication of whether the immune and stromal ratios are significant in patients with osteosarcoma is the relationship with survival, so we performed a Kaplan–Meier analysis of three kinds of scores, and not surprisingly, the scores correlated positively with survival Figures . To asseP < 0.05, Figures p=0.1, Figures p  >  0.05, Figures In addition to the survival rate, it is worth discussing whether these three kinds of scores are correlated with other clinical indicators such as age and gender. The results indicated that gender is significantly correlated with ImmuneScore and ESTIMATEScore in patients  are received by comparing two groups (low- and high-ImmuneScore patients), with the median value as the cut-off Figures . CorrespTo move forward a single step in exploring the latent mechanism, we worked with Cytoscape software to set up the PPI network in the STRING database. Based on previous report, we chose TREM2 for further study . AccordiTaking the fact that the levels of TREM2 expression have positive correlation with OS patient survival into consideration, these two groups are in comparison in GSEA. Hallmark and C7 sets of both demonstrated that the groups with high expression of TREM2 have observably more enrichment in immunity-related gene sets, suggesting immunity-related functions, such as the complement response, allograft rejection, IL6/JAK/STAT3 signaling, and acquired immunity are substantially more vibrant Figures . TherefoTo move forward a single step in confirming the relevance between TME and expression levels of TREM2. CIBERSORT methodology is utilized to acquire the immune subpopulation composition of tumor-infiltrating. The establishment of twenty-two kinds of immunity-related cell profiles is executed as follows , and theIn the current study, genes of the tumor microenvironment that related to the survival of OS patients from the TARGET database are what we attempted to appraise. TREM2 is appraised to be engaged in immunity-related activities. More significantly, a battery of research on bioinformatics revealed that TREM2 is a prognostic biomarker for osteosarcoma microenvironment remodeling.The tumor microenvironment played a pivotal part in tumorigenesis and its progression. It is of strategic meaning to detect the underlying therapeutic targets which can contribute to the remodeling and facilitating the transition of the tumor microenvironment from a developmental state to an inhibitory state.Numerous research studies had elucidated the significance of tumor microenvironment in tumorigenesis . In osteFor the last few years, tremendous progress was acquired in immunotherapy, and the inhibition of immune checkpoint inhibitors (ICIs) in OS made significant progress , 22. HowAs a dominating signaling hub of pathology-induced immunity, TREM2 catch the attention of the leading role of myeloid cells in various pathological processes which can mediate immunosuppression . Many maCIBERSORT methodology is applied to accomplish the analysis of the proportion of TICs and completed the composition of twenty-two profiles of immune cells. The results exhibited that macrophages accounted for the highest proportion in the TME of OS, especially M2 macrophages. The fraction of M2 macrophages in high expression groups of TREM2 is higher, which may have a relation to the immune-suppressive TME. In addition, CD8-positive T cells and follicular helper T cells have a positive correlation with the differential expression of TREM2. Plasma cells, naive CD4-positive T cells, and M0 macrophages have a negative correlation with the expression of TREM2. All results suggest that the differential expression of TREM2 is linked to the levels of immune cell infiltration and is a critical target for ameliorating the prognosis of OS. As an attractive biomarker for modulation of individual immunotherapy who are intractable to therapy of ICIs and have a TME rich in TAM, TREM2 is tightly associated with TAMs .Applying the ESTIMATE algorithm, functional enrichment analysis is applied to acquire the gene of the tumor microenvironment of OS in the TARGET database. TREM2 catches our eye as a potential prognostic biomarker for OS patients. What is of interest is that, although TREM2 may mediate the immunosuppressive tumor microenvironment through macrophage M2 polarization, its expression level has positive relevance with the overall survival time of osteosarcoma patients. Further research is indispensable to disclose the mechanism of regulating and exploit novel immunotherapeutic strategies.


One session of high energy extracorporeal shockwave therapy (ESWT) was found to improve the healing of anterior cruciate ligament (ACL) after reconstruction in animal and human studies. This study investigated the effects of three and six sessions of low energy ESWT on graft incorporation and knee functions post ACL reconstruction in humans.Thirty participants with ACL injuries were recruited and assigned equally into three groups with 10 participants per group (n=10). Participants in the control group received physiotherapy alone without low energy ESWT. Participants in the 3ESWT group underwent three sessions of low energy ESWT (one session per week) combined with physiotherapy, and participants in the 6ESWT group received six sessions of low energy ESWT (one session per week) combined with physiotherapy. However, five participants were lost to follow-up. Evaluations of graft incorporation of the tibial tunnel using magnetic resonance (MRI) and Lysholm score were carried out before ACL reconstruction and after six months post ACL reconstruction.The number of grafts with partial incorporation in the tibia tunnel in 6ESWT was significantly higher compared with the number of grafts with non-incorporation at six months post-operatively, X2  =5.44, p =0.02. However, there was no significant difference between frequencies of graft incorporation in tibia tunnel in the control and 3ESWT groups, X2  =3.57, p =0.06 and X2  =2.78, p =0.10, respectively at 6 months postoperatively. Lysholm scores were significantly higher at 6 months post ACL reconstruction compared to the baseline value for each group . However, there was no significant difference in the Lysholm score between each group .Six sessions of low energy ESWT improved graft incorporation in the tibial tunnel. Both three and six sessions of low energy of ESWT does not affect the knee function score at six months post ACL reconstruction. The mechanism of ACL injuries reported is due to the rotation of femur and tibia in opposite directions under full body weight. It happens when there is a sudden turning to change direction while running. Common symptoms of ACL injuries include pain, swelling, instability, and loss of function in severe cases2. As the consequence of ACL injury, athletes are adversely affected from training or competing4.Anterior cruciate ligament (ACL) plays an important role to prevent anterior translation5. ACL reconstruction surgery aims to restore knee functions by achieving the tightest possible repair so that the reconstruction of the torn ligament can be the perfect biological replacement7. Anterior cruciate ligament (ACL) reconstruction is commonly performed by repositioning of a free tendon graft either from the patella or hamstring tendon into a bone tunnel8. The junction between the graft tissue is also known as ‘osteotendinous’ junction, ‘enthesis’, or ‘insertion site’9. In the long term, an ACL rupture can cause further intra-articular damage like meniscal tears, cartilage defects, and osteoarthritis. It has been reported that twothirds of primarily conservative treated patients decided to have ACL reconstruction after rehabilitation10.Conservative or surgical treatment of knee instability is indicated for regaining pre-injury function6. Furthermore, the functional outcome of this surgery depends on the firm healing of graft incorporation with bone in the bone tunnel8. Some of the studies found that the graft incorporated well with the bone after implantation14 but other studies have shown contrasting results16. It is known that if the bone and tendon do not heal together, it may lead to future knee pathology. It has been estimated that 8-10% of ACL reconstructions result in recurrent instability and graft failure. Wang et al8 mentioned that two-third of the graft failed because of graft pull-out at 24 weeks post-surgery.A good ACL reconstruction outcome is said to have lesser knee laxity with a firm fixation1. In addition, it is claimed that the graft fixation using a hamstring tendon is less secure3. Different techniques and modalities are applied to accelerate the healing of new tendon implanted to the bone, however, inconsistent results have been reported20. Therefore, more studies that aim to reduce the risk of graft failure by increasing the rate of graft healing using external physical stimulus produced by the shockwave machine are warranted.One of the factors that affect the surgical outcome of ACL reconstruction is the rate of graft healing27. Sports injuries related to tendon-bone junction became an interest among the researchers due to its delay in healing for conservative treatment and increasing failure rate in surgical procedures. The complication of tendon and bone to blend is attributed to the non-homogenous structure in both types of tissues28. Although after a surgical intervention, the normal healing process of tendons is slow due to its low vascularity and low cellular composition in nature29. Thus, few strategies have been developed including using shockwave therapy as external mechanical stimulations to improve the natural tendon-bone healing.Several studies have demonstrated the effectiveness of extracorporeal shockwave therapy to treat various chronic musculoskeletal pathologies38. The mechanical stimuli produced by the shockwaves can induce physiological responses at the cellular level41. Several studies have previously reported the effectiveness of shockwave therapy on bone structure43 and tendon structure alone44.From the current literature, shockwaves therapy is one of the rehabilitation modalities that has the potential to be applied to a tendon-bone junction to accelerate the healing process46. Previous studies reported that extracorporeal shockwave therapy (ESWT) is beneficial to induce the ingrowth of neovascularization and improvement of blood supply at the bone-tendon junction31, promote tissue repair8, upregulation of angiogenic and osteogenic growth factors47 and increase cortical bone formation in acute fracture48. Additionally, it was also reported that ESWT is beneficial in promoting tendon-bone healing in the bone tunnel after ACL reconstruction in an animal8 and human study32. Nevertheless, a limited number of studies investigate the effects of ESWT on graft incorporation after ACL reconstruction.Besides, shockwave therapy is listed as one of the biological therapies for tendon injuries of the knee joint3. High Lysholm score and International Knee Documentation Committee (IKDC) score are among the factors that influence the recreational sports player’s return to sports at the same level49. To date, based on a systematic review finding46, only Wang et al32 reported the effectiveness of ESWT post ACL reconstruction on physical function. Lysholm score was found to be better in participants who received ESWT compared with the participants who did not receive ESWT after one and two years of follow-up post hamstring autograft ACL reconstruction. However, in the similar study, the authors reported that the IKDC score was not affected by the intervention prescribed32.The use of the Lysholm score as evaluation tool of knee functions has been reported to be more sensitive for the evaluation of activities of daily living and recreational or competitive sports46. Thus, additional studies are warranted to validate the previous study. This current study aimed to investigate the effects of three and six sessions of low energy ESWT on graft incorporation and knee functions in individual post ACL reconstruction.Based on a systematic review study, there is limited evidence regarding the effects of ESWT following arthroscopic knee surgeryIn this study, thirty participants with a single autograft hamstring reconstruction were recruited. However, twentyfive participants with a mean age of 26.8 ± 5.8 years old completed the present study. The participants were patients with an ACL tear, underwent primary single autograft hamstring ACL reconstruction, and the injury was caused mainly by involvement in sports activities. However, a few injuries were caused by other factors such as motor vehicle accidents and falls. Participants with primary single autograft patella tendon ACL reconstruction, revision ACL surgery, previous knee surgery which interfere with the knee functions, having multiple knee ligament injuries, medical problems, and participants on NSAIDs medication and/or consume calcium supplementation were excluded from this study.Ethical approval to conduct this study was obtained from the Human Research Ethics Committee of Universiti Sains Malaysia (JEPeM) (USM/JEPeM/16090303) and Medical Research and Ethics Committee (MREC) (NMRR-18-95341870 (IIR)). This study was also registered with the National Medical Research Register (NMRR). All the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 1983. This is a quasi-experimental study and uses an opportunistic sampling method. A day before the operation, all potential participants were fully informed by the researcher about the nature of the experiments, purposes of the study, procedures, termination or withdrawal, potential risks, and benefits of participating in this study. The participation of the participants in this study is voluntarily, and they were allowed to withdraw from this study at any time during the study. If they agree to participate, they were asked to fill in the participant information and consent form. After that, participants were screened to determine the inclusion and exclusion criteria are met. The qualified participants then proceed to the next phase of this study, which was a grouping into control, 3ESWT, and 6ESWT groups.Thirty male participants (N=30), with age ranging from 20 to 36 years old were recruited from the orthopaedic clinic of Hospital Universiti Sains Malaysia (HUSM) and Hospital Raja Perempuan Zainab II (HRPZ II) in Kelantan state of Malaysia. They were age matched and assigned into three groups using non-random method, i.e. natural experiment type of quasi-experimental design with 10 participants per group (n=10 per group). The participants have chosen to follow the normal protocol after ACL reconstruction (control group) or decided to try out the new therapy, i.e. shockwave therapy plus the normal protocol after ACL reconstruction (intervention groups) to suit with their convenience. Their preferable group was chosen based on their limitations such as transportation, location of their house and its distance from hospital, and commitment to attend all the scheduled follow-up sessions. The three groups were; i. Physiotherapy alone without ESWT control group (Control group), ii. Three sessions of low energy ESWT with one session per week combined with physiotherapy group (3ESWT group) and iii. Six sessions of the intervention of low energy ESWT with one session per week combined with physiotherapy group (6ESWT group).2.All ACL reconstruction procedures were carried out by an orthopaedic surgeon in HUSM and HRPZ II based on a standard procedure. Both surgeons are accredited to perform these procedures. Intra-operatively, all participants underwent examination under anaesthesia to confirm the diagnosis of ACL tears. The reconstruction surgeries were carried out arthroscopically. Semitendinosus and gracilis tendon grafts were harvested and whip stitch were sutured at both ends. The grafts were folded in half and tensioned for 20 minutes. The tibial and femoral tunnels were drilled via the standard technique. The grafts were secured to the femoral end with a suspensory cortical button and the tibial end were secured with a bioabsorbable screw31 i.e. 0.18 mJ/mm2 = 14kV for the 6ESWT group, similar total energy used by Wang et al30, Wang et al8, and Pavone et al50. While, half of the dosage, i.e. 0.09 mJ/mm2 was used for the 3ESWT group. It was stated in Rompe et al51 and Speed52 that ESWT is considered low energy with energy flux density (EFD) ranging from 0.08 – 0.27 mJ/mm2, medium energy with EFD ranged from 0.28 – 0.59 mJ/mm2, and high energy with EFD more than 0.60 mJ/mm2. ESWT was administered at 500 shocks, once per week for either 3 or 6 weeks. The frequency of treatment was selected based on previous studies56. After the intervention, the treatment area was inspected again to observe if any redness or hematoma occurred. Participants in the control group did not receive ESWT after ACL reconstruction.The application of ESWT was administered to the participants once a week for three weeks  in the 3ESWT group. Meanwhile, for the 6ESWT group, the application of ESWT was administered to the participants once a week for six weeks (within week 7 until week 12 post ACL reconstruction). The ESWT was generated from a shockwave therapy device . The treatment area was localised by palpation. The applicator of the ESWT was put directly on the skin region, perpendicular to the femoral tunnel at the lateral side of the knee joint, directed latero-medially, after applying the ultrasound gel. Then, shockwaves were released as the applicator button was pressed. The therapy was performed without anaesthesia. The total energy used was adapted from a study done by Wang, Huang and Pai58, since this was in the post-operative stage, the structures were mainly bone in nature, and the pain tolerance was low. The total number of shocks i.e. 500 shocks was similar to a previous animal study investigating the effect of the shockwave on bone healing around metal implants8.This study aimed to deliver low energy of shockwave without anaesthesia but within pain tolerance for a few sessions of interventions. Based on the recommendation from the manufacturer of shockwave machine  and previous practice reported in the literature, one session of shockwave therapy per week for three and six weeks was scheduled. We decided to use the minimal tolerable intensity for our participants which was 1.5 bar59 as a guideline. There might be a slight modification of the standard protocol based on the capability and condition of the patients. Thus, the standard ACL reconstruction rehabilitation protocol used in this current study was the ‘modified accelerated rehabilitation program’ based on a protocol by Shelbourne and Nitz59.The normal physiotherapy rehabilitation was performed by all the participants in all groups including continued wearing of knee braces and crutches post-operatively. All the participants were required to follow a physiotherapy regime prescribed by medical doctors and physiotherapists based on a standard ACL reconstruction rehabilitation protocol. The rehabilitation protocol was standardised by using an ‘accelerated rehabilitation program’ adapted from Shelbourne and Nitz60. In a clinical setting, MRI was considered to be the best method in the evaluation of the healing of tendon to bone at the tendon-bone interface after ACL reconstruction in human subjects61. To determine the participants’ knee functions, Lysholm score were collected at pre-operatively (T0) and six months post ACL reconstruction (T4). Participants were asked to respond to the questions. The total scores would indicate the outcome of the ACL reconstruction, and they could be classified as excellent (95100), good (84-94), fair (65-83), and poor (≤64)62.Graft incorporation was determined by the quality of the bone-tendon interface and the signal quality of the ligament itself which can be observed on the image of Magnetic Resonance Imaging (MRI) at six months post ACL reconstruction. The result obtained from this evaluation determined if the treatment could accelerate the healing process of the new graft to bone six months post-operatively. The participants were screened after completing the screening forms which contained a few questions related to health status to ensure that they were safe and eligible to undergo MRI screening. The device used for this imaging was the Philips MRI machine . The participants were asked to lie down on the MRI table. The affected knee was slightly bend at around 15° as the knee coil was placed at the joint. The total duration of this scan was around 40 to 45 minutes each session. The participants were given the MRI radiographer alert button during the scanning session. MRI image evaluation was performed by a trained and experienced musculoskeletal radiologist. He was blinded from the hypothesis of the study, participant’s group, and participant’s knee joint functional score. The images were coded and information that can identify each participant was removed. Only the research staffs of this study were able to link the code with each participant. The graft incorporation within the tunnels was characterised as ‘complete’, ‘incomplete’, or ‘none’. For complete incorporation, the bone-tendon interface was mature. For incomplete incorporation, the bone-tendon interface was partly mature and partly immature, while, for no incorporation, no evidence of a mature bone-tendon interface was foundStatistical Package for Social Sciences (SPSS) version 26.0 was used for the statistical analysis. All the data are presented as mean ± standard deviation (SD). Mixed ANOVA was performed to determine the significant differences within and between groups. A ‘p’ value of <0.05 was considered statistically significant and was used for all the comparisons. If the mixed ANOVA test showed a significant difference, the post hoc test using the Bonferroni correction test was used to analyse the differences between each measurement. If the Bonferroni correction test showed a significant difference, mixed ANOVA test with the selected cases was used to analyse the differences between each measurement within each group. One way ANOVA with contrast test was used to determine the differences between the control group and the intervention groups, and the differences between both intervention groups. A Chi-square test for differences was performed to determine the significant differences for categorical data.The participants’ demographic characteristics are presented in The result showed that the number of graft with partial incorporation in the tibia tunnel in the 6ESWT group was significantly higher compared with the number of the graft with non-incorporation at 6 months post-operatively, X2  =5.44, p =0.02 . Howeverp = 0.247. In addition, there was no significant main effect of group observed, F  = 2.798, p = 0.083. However, there was a significant main effect of time in Lysholm score, F  = 74.41, p<0.001. Post hoc test using the Bonferroni correction revealed that there were significant differences in a comparison of time for all three groups; control , 3ESWT , and 6ESWT , p=0.002, respectively  = 1.49, ectively . The Lys35, increases regeneration of the fibrocartilage zone36, enhances bone remodelling in the delayed tendon-bone junction33, induces bone formation and healing of segmental femur defects associated with bone morphogenetic protein expression in callus63, enhances bone mass and bone strength after fracture64, induces neovascularization and improves blood supply at the tendonbone junction31, increases the number of trabecular bone around the tendons, and improves the contact between bone and tendon8, enhances the tensile strength of the tendon-bone interface35 and improves bone mineral status33.To date, most human studies have reported the clinical effects of ESWT, meanwhile most animal studies reported the cellular effects of ESWT. ESWT has been speculated to accelerate the healing of transplanted tendon to bone via a few mechanisms. In animal studies, it was found that shockwave promotes osteogenesiset al8 who applied one session of low energy of ESWT on tendon-bone junction after ACL reconstruction surgery in rabbits. They found that there was significantly more trabecular bone around the tendon, better incorporation of the tendon to bone, and higher tensile strength in the intervention group compared to the control group. Nevertheless, similar modes of graft failure were noted between the two groups. In other animal studies, high energy shockwave therapy has been reported to promote new bone formation34, higher expressions of VEGF34 and regeneration of the fibrocartilage zone36 in the delayed tendon-bone junction healing after partial patellectomy compared to the control group. Qin et al34 found that scar tissues were formed at the tendon-bone junction healing site in the control group after eight weeks postoperatively. Additionally, Wang et al33 found that there was significantly higher bone mineral status, tensile load and strength in the intervention group compared to the control group.The effects of low energy of ESWT on tendon-bone junction in animal was studied by Wang et al35 showed that low energy shockwave therapy was as effective as high energy shockwave therapy in the treatment of delayed tendon-bone junction healing after partial patellectomy. They found that both intervention groups have larger bone areas, greater new bone volume, and significantly higher failure load at the tendon-bone healing junction than the control group at week 12 post-intervention. The results of these abovementioned studies were based on small animal i.e. rats/rabbits/dogs. The anatomy and physiology of the knee in those animals may not necessarily resemble the human subject. Therefore, the effects of ESWT in human subjects may be different from animals.A comparison study by Chow 67. Based on systematic reviews46, only one study used Magnetic Resonance Imaging (MRI) to investigate the effect of ESWT on tibial tunnel enlargement diameter after ACL reconstruction in humans32. In their study, high energy shockwave was applied once immediately after the operation with the same anaesthesia by focus type shockwave. Both diagnostic methods, i.e. radiograph and MRI presented a smaller tibia tunnel after two years and significantly decreased in tibia tunnel enlargement after six months and two consecutive years post-operatively. Based on the outcome, ESWT has the potential to promote tendon-bone junction healing at two years post ACL reconstruction surgery.Several previous studies have provided evidence that few sessions of shockwave therapy might accelerate the enthesis healing by reducing the pain level, improved functional activities, including the participants’ quality of lifeHowever, to date, studies using radio-imaging such as MRI to show the progress of graft incorporation healing especially after ACL reconstruction in humans are still lacking. Therefore, the present study was carried out. The main finding of the current study was that the number of the graft with partial incorporation in the tibia tunnel was significantly higher compared with the number of the graft with nonincorporation at six months post-operatively in the 6ESWT group. The current study also found that the number of the graft with partial incorporation in the tibia tunnel was higher compared with the number of the graft with nonincorporation at six months post-operatively in the control and 3ESWT group, respectively, however, it was not significant statistically. These findings showed that six sessions of low energy of ESWT may be effective to accelerate the graft healing by inducing graft incorporation post ACL reconstruction in humans. These findings also showed that graft incorporation of each participant responded differently towards the ESWT intervention and the number of ESWT sessions. Thus, it was speculated that the mechanism of ESWT on graft incorporation in humans may be similar with the mechanism in the animal studies.68, which was carried out to compare the effects of two different dosages of radial shockwave therapy (rESWT) on Jumper’s knee. The participants in the intervention group received 1000 shocks of rESWT with 2.0 bar intensity, and 20Hz frequency and were applied twice a week for 4 weeks. Meanwhile, the participants in the control group received 2000 shocks of rESWT with 2.0 bar intensity and 20Hz frequency were applied once a week for 4 weeks by using the same device. That study showed that physical functions were improved in both control and intervention groups but not significantly different. However, the increment of physical function score was significantly notable in the experimental group at 4th week compared to 3rd week post-intervention.This present study also found that knee function scores were significantly improved in the control, 3ESWT, and 6ESWT groups at 6 months post-operatively compared with the knee function scores at baseline, p=0.002, respectively. However, there was no significant difference in the Lysholm score between each group . Similar findings were observed in a previous study by Mohamed and Gaddur69 reported that a combination of three sessions of ESWT with rehabilitation in patients with chronic plantar fasciitis was effective in improving the local function of foot and ankle but not the global function after six weeks, three months, and six months post shockwave therapy compared with baseline value. The energy dose was varied and specific to the individual, and different between sessions as the researchers applied ‘maximal comfortably tolerated’ energy dose for each participant.Wheeler70. In another study, it has been found that the effects of ESWT on functional outcomes among participants with knee osteoarthritis were dose-dependent. Both low and medium energy ESWT improved knee function compared with the baseline value. However, at week 1, 4, and 12, greater improvement was found in the medium energy group compared with the low energy group71. Nevertheless, there was no control group enrolled in these previous studies.Contradict with the findings from the present study, some researchers found that ESWT contributes to the improvement of the physical function of the participants. The physical ability of participants with calcific tendonitis of the shoulder, chronic Achilles tendinopathy, and lateral epicondylitis was improved compared to baseline value at one-year post ESWT72. In a comparison study, 5 sessions of shockwave therapy resulted in an improvement in physical function in the WOMAC sub-score among participants with knee osteoarthritis at week 5 post-intervention compared to the kinesiotherapy group. The shockwave therapy session was carried out once a week for five weeks with one-week interval73. In a randomised control trial study, it was found that shockwave therapy was more effective than ultrasound therapy in improving the physical function among participants with osteoarthritis knee. The ESWT group received 1000 pulses during the first treatment, 1500 during the second and the third treatments, and 2000 during the fourth and the fifth treatments, respectively . The participants attended five sessions of shockwave therapy, i.e. one session per week for five weeks with weekly intervals. The researchers used Knee injury and Osteoarthritis Outcome Score (KOOS) as a primary outcome measure to assess physical function among participants74. These studies showed that ESWT was superior to laser, kinesiotherapy, and ultrasound in improving physical function among participants with osteoarthritic knee.A few comparative studies have been done to investigate the effectiveness of ESWT on physical function among participants with knee osteoarthritis. In a retrospective study, radial ESWT was effective in relieving symptoms of knee OA, including improving knee functions which was evaluated using total WOMAC score. After 5 sessions of ESWT with 3 days interval, ESWT showed greater beneficial effects compared with laser therapy at week 6 and week 12 post-therapy75.A randomised controlled trial study found that four sessions of a low dose ESWT, i.e. once per week for four consecutive weeks significantly improved the physical function level of participants with the osteoarthritic knee at week 5 and week 12 post-shockwave therapy in the intervention group compared with the control group. The participants in the intervention group received a total of 2000 pulses of 8Hz frequency at 2.5 bars while the participants in the control group received sham shockwave therapy with a similar protocol of ESWT but using 0.2 bars of air pressure76. In a long term follow-up study, three sessions of low energy radial ESWT improved the functional outcome of the participants with insertional and non-insertional Achilles tendinopathy after two years post-intervention. The researchers applied 2500 pulses per treatment and were administered at weekly intervals. The ESWT dosages were 1.5 bar with 10Hz for the first 500 pulses and were increased to 2.5 bar for the remaining of 2000 pulses77. Most of the previous studies reported the effects of ESWT on physical function among participants with chronic diseases such as jumper’s knee, chronic plantar fasciitis, osteoarthritic knee, calcific tendonitis of the shoulder, chronic Achilles tendinopathy, and lateral epicondylitis. The enhancement in physical function can be seen as early as one-week post-ESWT in these previous studies.A meta-analysis study concluded that the physical function of participants with osteoarthritis knee using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was significantly improved at 4 weeks, 8 weeks, and 12 weeks post low-intensity shockwave therapy compared to the control groupet al32 showed that ESWT was effective in improving participants’ functional score at one and two years post-operatively. It was postulated that variation in conditions treated, different methods of ESWT application, dosage or energy used, number of ESWT sessions, type of shockwave machine, and duration of follow-up period may lead to inconsistent results. Besides, it was speculated that improvement in physical function following ESWT post-surgical procedure can only be observed after a longer follow-up period. The outcome of physical function may be influenced by patients’ selfmotivation and adherence to the rehabilitation sessions as the confounding factors.There is limited evidence on the effectiveness of ESWT on local function or global physical function among participants after surgical procedure. Wang There are a few limitations in this current study. The participants in the present study were not randomly selected and allocated into groups as limited numbers of patients met the inclusion criteria and not many of them agreed to participate in this study. Besides, the commitment, transportation, and logistics problems are the factors that need to be considered when allocating them into control or the intervention groups. Thus, to fulfil the required number of patients in the study period, randomisation cannot be done. Due to the aforementioned problems, the sample size for this study was relatively small. Nevertheless, the sample size calculation has been performed based on a previous related study. In addition, the duration of follow-up was relatively short. Thus, future studies with long term followup and randomisation with large sample size is warranted to confirm and validate these findings.As a conclusion, six sessions of low energy ESWT improved graft incorporation in the tibial tunnel. Both three and six sessions of low energy ESWT did not elicit additional benefits on knee function score at 6 months post ACL reconstruction.


The aim of this multicenter clinical study was to examine the oral health-related quality of life and oral hygiene in adolescents before and during aligner therapy.Forty subjects  scheduled for aligner therapy  were given oral health-related quality of life questionnaires, Oral Health Impact Profile (OHIP-G14) and Psychosocial Impact of Dental Aesthetic Questionnaire (PIDAQ), to complete within their treatment . To assess oral hygiene, a questionnaire to take home was used, and plaque level was evaluated with the Quigley-Hein Plaque Index (TMQH) modified by Turesky et al.The OHIP-G14 mean score before aligner therapy was 3.3 ± 3.2, and 4.9 ± 5.4 after 1 year. The PIDAQ showed a positive psychological change in the well-being, as well as a more effective at-home oral hygiene regime. On average, the TMQH remained at a low level (grade 2 of 0–5). The initial insertion of the aligners caused the most significant changes in all parameters (except TMQH).Oral health-related quality of life is only slightly affected during the first year of aligner therapy in adolescents. Oral hygiene at home is intensified and there is no increased dental plaque accumulation.Dentofacial esthetics is a subset of the so-called Oral H-Related Quality of Life (OHRQoL) which should be considered more during orthodontic therapy. It is becoming increasingly common for patients to select orthodontic therapies based solely on esthetic considerations –4. The iThe desire for an esthetically pleasing orofacial appearance and an increased quality of life associated with it results in a growing interest in low-profile orthodontic appliances for the regulation of tooth and jaw malocclusions –10. RemoChildren and adolescents represent the main group of patients in orthodontics. During this age range, the periodontium can still be significantly remodeled while making use of patients’ physiological growth . In thisThe objective of this multicenter clinical study was to examine the effect of aligner therapy in a cohort of adolescents  on 1.) oral health-related quality of life and 2.) oral hygiene during the first year of treatment.The presented results are published as an exploratory analysis of 1-year results of a larger ongoing clinical study “Studie zur Untersuchung des Mundhygienestatus und der Lebensqualität des Patienten vor, während und nach der Aligner-Therapie .” The primary endpoints are OHIP and PIDAQ at 1 year after debonding and will be reported after completion of the study.t-tests. This required 45 participants. Assuming a dropout rate of about 18%, we included 55 patients.The clinical surveys were performed in the Department of Orthodontics of the University Medical Center at the Johannes Gutenberg-University Mainz and the orthodontic practice of Dr. med. dent. Thomas Drechsler in Wiesbaden. The focus of the larger study, for which sample size calculation was performed using SAS PROC POWER, was to establish a medium effect in three primary endpoints, i.e., an effect size of 0.5 at the 1.67% significance level each (Bonferroni-correction) with 80% power using three paired So far, 1-year results are available for 40 adolescents scheduled for orthodontic therapy with aligners   who were 11 to 17 years old at the time of study start. Subjects and their legal guardians received written information regarding the study procedure and signed informed consent forms prior to study entry. Participants were required to agree not to participate in any other clinical studies for the entire data collection period. They were instructed to have no external professional dental cleanings, to maintain their current brushing habits , and to not clean their teeth the morning prior to the study appointments. The inclusion criteria were 1) general health, 2) at least 16 natural teeth—at least 8 of which were anterior teeth—and 3) vestibular and lingual tooth surfaces sufficiently assessable. Exclusion criteria were 1) lack of indication for orthodontic therapy, 2) expected insufficient compliance, 3) other ongoing orthodontic treatment, 4) previous therapy with multibracket appliance, 5) presence of severe periodontal disease, 6) ongoing periodontal therapy, 7) allergies to dyes or food, 8) more than three carious defects requiring treatment, 9) use of an antibiotic within two weeks prior to study entry, 10) professional dental cleaning within two weeks prior to study entry, 11) having a pacemaker, 12) pregnancy, and 13) syndromic conditions.Two valid and reliable questionnaires were used to assess oral health-related quality of life: Oral Health Impact Profile (OHIP) and Psychosocial Impact of Dental Aesthetic Questionnaire (PIDAQ) , 28. TheThe surveys were distributed over the first year of aligner therapy: divided into five visits . The study sessions preceded each orthodontic appointment. Due to the limited timeframe of 1 year, the visits after the study such as further treatment time, debonding, and follow-ups after debonding are not included in this data. The data collected in visit 1 provided baseline values. A one-time oral hygiene instruction with illustrations was presented during the first visit. At the beginning of each visit, participants were given the questionnaires to complete. This was then always followed by the clinical survey of the TMQH with subsequent professional tooth cleaning.p values should be interpreted in a descriptive fashion. In this exploratory analysis, no adjustment for multiple testing is performed. p values less than or equal to 0.05 are termed significant.The following software was used for statistical analysis: SAS 9.4 , IBM SPSS 23 , and Microsoft® Excel. Mean imputation was used for incomplete data. Descriptive statistics were drawn from the data. Differences between time points were assessed using Friedman’s test . These tThe collective included a total of 40 adolescents , aged 11 to 17 years (Ø 13.6 years) at the study baseline , though that of the female population is slightly higher than that of the males  and the TMQH (p = 0.441). However, differences can be established in the case of the PIDAQ (p < 0.001).The TMQH documents relatively constant mean index values across visits Table . The FriThis multicenter clinical study aimed to assess the effects of aligner therapy  on oral health-related quality of life and oral hygiene over the first year of therapy in adolescents. The population was divided into two groups of approximately equal size, 18 female and 22 male subjects. The study included subjects between the ages of 11 and 17 years at the start of therapy, which represents the majority of the orthodontic patient clientele, and yet, this age group has received little attention in previous studies regarding the topic of this study.Studies on oral health-related quality of life, which compare treatments with aligners to those with fixed appliances, conclude a higher quality of life with aligner therapy , 26, 37.For this study, the German short version of the OHIP, the so called OHIP-G14, was used. This version is based on the original version by Slade  and its Critically, it can be questioned why, in the context of the study conducted here on children and adolescents, the specific questionnaires available were not chosen to assess the OHRQoL. The most widely used questionnaire for children and adolescents is the Child Perceptions Questionnaire, which is characterized by the highest validity and reliability , or the Even though available in the versions, for ages 8 to 10 and 11 to 14 , 42 and The OHIP is one of the most widely used instruments for measuring OHRQoL in Europe . FurtherWhen regarding this scientific work, the evaluation of the OHIP-G14 reveals OHRQoL limitations within the first year of therapy. The most significant restriction of the OHRQoL occurs after the initial insertion of the aligners in the period from visit 1 to visit 2. A decreasing trend is seen in the following visits. After 1 year, the OHRQoL was only slightly reduced in comparison to the study baseline value. Further studies support the conclusion that OHRQoL experiences the greatest limitations at the beginning of orthodontic therapy , 50. PaeFor the present study, it was relevant to choose two kinds of measurement instruments with different focus to elicit OHRQoL. This was to include a wide range of aspects of OHRQoL. The OHIP is also used in general dentistry, whereas the PIDAQ was developed specifically for the survey in orthodontics. The German short version of the OHIP with 14 questions (OHIP-G14) and the PIDAQ in a modified version for children and adolescents were used. The two questionnaires for the assessment of OHRQoL complement each other in terms of content. The OHIP measures the social influence of oral diseases or, in the case of orthodontics, dysgnathic dentition, while the PIDAQ measures the psychosocial effects of dental esthetic appearance.The data obtained with the PIDAQ makes it clear that the OHRQoL of the subjects is positively influenced over the course of the first year of therapy. This is particularly true for the male subjects since they have continuously lower mean values than the female subjects. After insertion of the aligners, an increase in psychosocial well-being can be observed across genders. Initially, this seems to contrast with the results of the OHIP-G14; here, the greatest restrictions are indicated after insertion of the aligners. The contradicting results can be explained by the prospect of an esthetically pleasing orofacial appearance. Studies of subjects undergoing orthodontic therapy with fixed multibracket appliances also find an increase in psychosocial well-being based on the PIDAQ , 51, 52.With regard to oral hygiene at home, the test subjects stated that they were more conscientious about it. This is particularly evident in the period from visit 1 to visit 2 and is more noticeable in the male subjects than in the female subjects. The instructions on oral hygiene at home and the professional dental cleanings performed during each visit may have been motivational in this respect. These measures may have contributed to an increased awareness of the importance of home oral hygiene. The Friedman test provides statistical evidence for the reported intensification of oral hygiene at home. Other studies also state improved oral hygiene under orthodontic therapy, both with aligners  and withThe clinical survey on dental plaque accumulation using TMQH provides relatively constant values over the entire survey period. These lie in the lower range of the index—at grade 2 (range 0–5). No significant gender-specific differences are concluded. The Friedman test confirms that there are no statistically verifiable differences between the survey periods. Other studies do conclude increased plaque levels in subjects undergoing therapy with fixed orthodontic multiband appliances . StudiesOverall, it can be concluded that oral health-related quality of life does not experience statistically provable limitations in adolescents (11 to 17 years) in the first year of therapy with aligners . Psychosocial well-being increases throughout the course of the treatment. Oral hygiene at home is intensified and there is no clinical evidence of increased dental plaque accumulation. With minor restrictions in the quality of life and increased psychosocial well-being, combined with the prospect of an esthetically pleasing orofacial appearance, sufficient compliance can be assumed. Especially considering the before mentioned aspects and the increased demand of orthodontic treatment in this age range, a therapy with aligners —as a less conspicuous orthodontic appliance—should be considered for the central orthodontic patient clientele. This is also supported by the wide range of indications for this treatment method.


For decades, the distant progression of breast cancer has been the purview of systemic therapy alone or with low to moderate-dose radiation therapy intended for the palliation of symptomatic metastases. However, for decades there have been anecdotes of long-term disease-free survival with more aggressive local treatment of one or more metastases. The hypothesis of oligometastases is that the treatment of a clinically limited number of distant metastases can change the natural history of stage IV breast cancer. The advance in the technology of stereotactic body radiation (SBRT) has made it more possible to offer a non-invasive, yet potentially disease-modifying, metastases-directed ablative treatment in place of surgery or a palliative radiation regimen. Although there are promising local control and survival outcomes in phase I/II trials, there is still a lack of phase III evidence of ablative SBRT results showing any change in the natural history of metastatic breast cancer. Limited oligometastases may call for an ablative approach with SBRT when definitive long-term local control is needed for the best palliation against symptomatic progression in challenging locations. Some oligometastases that have progression on a certain systemic regimen, while others remain stable or in remission, may also be treated with SBRT in the hopes of prolonging the use of that regimen. Whether SBRT should represent the standard management for stage IV breast cancer of a limited number or of limited progression requires confirmation by phase III data. This review will discuss the data from key clinical trials as it applies to decision making in typical clinical cases considered for potentially ablative SBRT for oligometastases or oligoprogression. The systemic hypothesis by Fisher had been the dominant model explaining the natural history of breast cancer and stage IV disease for 30 years, from approximately 1985–2015 . Th. Th13]. Based on this randomized phase II data, SBRT to the oligometastasis in her sternal lesion offered the hope for improved progression-free and overall survival compared to systemic therapy with or without palliative radiation alone. One in five patients may be alive without progression or new metastases >5 years. In addition, SBRT offers improved durable long-term control, if not out-right total ablation, of the oligometastasis compared to only the observation or palliative treatment of it. The phase III data are pending completion and reporting of the follow-up SABR-COMET-3 and SABR-COMET-10 studies ,18.In the previous case, there was not a complete response to neoadjuvant chemotherapy in the local-regional disease or in the sternum. However, what is the role of SBRT if an oligometastasis is more responsive to systemic therapy? n = 65) versus systemic therapy plus metastasis-directed therapy (n = 60). The oligometastases in the 60 patients assigned to the ablative treatment arm received SBRT in 56 patients (93%), surgery in 1 patient (2%), and no protocol therapy in 3 (5%). Furthermore, 78% had metachronous and 22% had synchronous oligometastases. After a median follow-up of 35 months, the mean progression-free survival (PFS) was 23 months versus 19.5 months, and 3-year PFS 33% versus 38% (p = 0.36). The 3-year OS was 72% versus 69% (p = 0.54). There was no difference in the appearance of new metastases outside the treated oligometastases (40%), but there was a lower rate of progression within the oligometastases treated by SBRT (7% versus 29%). There was a high quality assurance and pre-review of the treatment plans and no grade 5 toxicity. Based on the phase II analysis, the subsequent planned phase 3 trial enrollment was cancelled.There are data to suggest that the observation of these oligometastases was the best approach in this woman, with a good response to systemic therapy. The NRG BR002 trial was a randomized phase II trial exclusively for breast cancer patients with 1–4 extracranial metastases, controlled local-regional disease, and no clinical progression for up to 12 months on systemic therapy  14]. Ov. Ov14]. Therefore, in this patient with lung metastases responding well to systemic therapy, observation without SBRT directed to each of the three lung metastases was appropriate. The patient was spared any risk for toxicity from surgery or SBRT, and the results of the NRG BR002 do not support a hypothesis that metastasis-directed therapy could reduce the incidence of other distant metastases. The three oligometastases remain in remission, but if there was a detectable local recurrence in one or more of them by CT surveillance, then SBRT for local control could be considered in the future.The majority of the patients in the BR002 trial had metachronous oligometastases after the initial local-regional treatment. In this case, the two oligometastatic lesions in the liver were metachronous from the primary breast cancer and occurred on her first-line adjuvant endocrine therapy. One option for management could have been a change in her systemic therapy alone, from tamoxifen to an aromatase inhibitor with a CDK 4/6 inhibitor. The data from the SABR-COMET and NRG BR002 phase II trials are contradictory in terms of whether there would be an improved PFS and OS with SBRT. The National Health Service England conducted a large prospective registry of SBRT for metachronous 1–3 oligometastases  15]. Pa. Pa15]. Cases 1–3 have been of synchronous or metachronous oligometastases. In a case of oligoprogression, there can be one or more limited number of sites of metastases that have not responded or progressed on a systemic regimen, while the others remain under control. p = 0.001), but not the breast cancer cohort . Based on these data, SBRT may not be associated with the change in the natural history of her metastatic breast cancer.In this case of extensive polymetastases, one option for the management of stage IV breast cancer that progressed on systemic therapy would be to change the systemic therapy. The Consolidative Use of Radiotherapy to Block oligoprogression (CURB) trial randomized patients with breast or lung cancer with up to five extracranial sites to the standard of care systemic therapy and SBRT up-front or delayed at further progression  16]. Ov. Ov16]. However, the option of SBRT to this one area of oligoprogression in the cervical spine could allow her to remain on her current, well-tolerated systemic therapy, which is controlling her other polymetastases, and avoid a change to cytotoxic systemic therapy. More importantly, the consequences of local progression in this location of the cervical spine are potentially severe. Failure to achieve local control at C1 could cause severe morbidity from fracture or surgery, immobilization, a decline in performance status, paralysis or even death. In this case, local control is essential to preserving not only quality of life, but survival itself.n = 115) or stereotactic body radiotherapy (n = 114). At three months, 40 (35%) of the 114 patients in the stereotactic body radiotherapy group, and 16 (14%) of the 115 patients in the conventional external beam radiotherapy group had a complete response for pain (p = 0·0002). With a longer follow-up in a subgroup of 137 patients, the risk for local failure was 15% for SBRT vs. 36% for palliative EBRT at two years (p < 0.001) [p-0.002). There were grade 3 vertebral compression fractures associated with SBRT in five patients (8%), but this was comparable to the rate of fractures from the progression of disease after conventional palliative radiation.The SC24 phase II/III trial randomized patients with painful spinal metastases to SBRT or moderate-dose palliative external beam radiation . Treatme< 0.001) . The neeTherefore, for this case of oligoprogression in a critical cervical vertebral location, SBRT offers the best long-term palliation at C1 by offering the best local control compared to low-moderate dose palliative external beam radiation. Whether SBRT, in cases of breast cancer oligoprogression such as this, alters the natural history of PFS elsewhere in other metastatic locations or changes the OS is unproven in the phase III data, but is of less concern than having durable local control in this cervical spine location.This review of SBRT for oligometastases and oligoprogression in stage IV breast cancer discussed several key clinical trials used for medical decision making in the four case examples presented . AdditioFor decades, the treatment of high-risk local-regional disease had the same uncertain status in cases of node positive or locally advanced breast cancer. In inflammatory breast cancer, with a survival benefit or not, PMRT is necessary for the prevention of morbid and even life-threatening local-regional progression. In a similar way, even if SBRT is “just” for better local control than standard external beam palliative radiation, for many patients, this more effective local control may actually be the most effective palliation and mitigates the risk of re-irradiation or other therapy for local control. The effective palliation of metastases in critical locations such as the spine, weight-bearing appendicular bones or critical organs can be associated with morbidity and mortality if not effectively treated by radiation, particularly in the setting of progression on systemic therapy. Clinical judgement is needed to determine when the greater local control benefit of SBRT is outweighed by the low risk for toxicity from SBRT. Whether a survival benefit to metastasis-directed SBRT in stage IV breast cancer exists remains to be determined by more phase III trials in breast-specific patient populations.