The application of laparoscopic surgeries has been increasing, and various uterine diseases in addition to leiomyoma/adenomyoma have become indications for total laparoscopic hysterectomy (TLH). Therefore, data acquisition and analysis of TLH procedures, including TLH for rare uterine diseases, have become important for improving surgical procedures and patient selection. To determine the prevalence of and risk factors for the occurrence of intraoperative and postoperative complications of TLH, we performed a multivariate analysis of the records in our hospital.We retrospectively reviewed the medical records of 323 patients who underwent TLH for the treatment of leiomyoma/adenomyoma (278 cases), low-grade (pre)malignant uterine tumours (40 cases) and other rare uterine diseases (5 cases) from January 1, 2015, to December 31, 2019. Of the rare uterine diseases, one case of caesarean scar pregnancy for which TLH was performed is introduced as a case report. To assess the effects of 11 representative factors, including patient characteristics, uterus and leiomyoma sizes, indications for TLH and others, we performed a multivariate logistic regression analysis.Among the 323 cases, 20 intraoperative complications and 15 postoperative complications were reported. In the multivariate analysis, “ovarian tumour” and “heavy uterus” were positively associated and “nulliparity” was negatively associated with intraoperative complications. There were no significant risk factors for postoperative complications. The only risk factor for operative complications directly related to the resected uterus was “heavy uterus”. Therefore, we could perform TLH relatively safely for patients with other indications besides leiomyoma/adenomyoma.Considering the factors detected in this analysis, the indications for TLH may be expanded. Owing to the increase in TLH for indications other than leiomyoma/adenomyoma, a more accurate determination of the treatment approach can be achieved. With the widespread application of total laparoscopic hysterectomy (TLH), the number of indications in addition to leiomyoma/adenomyoma has increased -3. The aHere, we present one rare case in which we performed TLH to successively treat caesarean scar pregnancy to illustrate our approach for treating a rare uterine disease. Simultaneously, we will introduce a proposed improvement for TLH that involves transvaginal morcellation of the uterus without a power device. Since the rates of indications besides leiomyoma/adenomyoma have increased annually with the increase of TLH cases, the safety verification of TLH has become more important. By analysing all the cases in our hospital, we elucidated factors that predicted the possibility of surgical complications.P < 0.05 was considered statistically significant.This study was reviewed and approved by the Human Ethical Committee of the University of Teikyo Hospital . The de-identified medical records of 323 female patients who underwent TLH from June 1, 2015 to December 31, 2019 were reviewed retrospectively. In these cases, bilateral salpingectomy (BS) or BSO was performed during TLH. This study also included 25 cases with concomitant PLA and 19 cases with concomitant LC, including 2 bilateral LCs and 17 unilateral LCs. Among the 323 cases, we extracted representative patients’ characteristics and surgical complications as described below. Statistical analyses were performed using JMP version 12 for Windows  to determine the correlations between patient characteristics and surgical complications. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to determine the strengths of the correlations. The patient characteristics (shown in Table 2); (4) “gynaecological surgical history”; (5) “concomitant procedure”, defined as concomitant PLA or LC; (6) “other indications”, defined as TLH for indications besides leiomyoma or adenomyoma; (7) “large leiomyoma”, defined as a dominant leiomyoma ≥ 8 cm as determined by magnetic resonance imaging (MRI); (8) “ovarian tumour”, defined as benign ovarian tumours; (9) “large uterus”, defined as the average uterus length ≥ 10 cm as determined by transvaginal ultrasound (TVUS) immediately before surgery; (10) “heavy uterus”, defined as a resected uterus weighing ≥ 500 g; and (11) “abdominal adhesion”, defined as abdominal adhesion detected by laparoscopic inspection immediately after the start of surgery. The criteria for “large leiomyoma” and “heavy uterus” were determined based on previous reports [To control for confounding factors, we divided the patients into two groups according to the presence or absence of each factor and performed a multivariate logistic regression analysis. In the 323 patients who underwent TLH, we assessed the influence of the following 11 factors: (1) “advanced age”, defined as an age ≥ 50; (2) “nulliparity”, defined as no previous delivery; (3) “high BMI”, defined as a body mass index (BMI) ≥ 25 , one spontaneous vaginal delivery (7 years ago) and a second caesarean delivery (5 years ago). Nine months before visiting our hospital, surgical abortion was performed.A 35-year-old Japanese woman, gravida 6, para 3, with 2 previous caesarean deliveries, was referred to our hospital at 6 weeks and 3 days gestational age for suspected abnormal placentation. She had a BMI of 25.9 kg/mAt presentation, TVUS revealed an 18-mm gestational sac (GS), a clear foetal heart-beat (FHB) and a crown-rump length (CRL) of 6.4 mm. The position of the GS was nearer to the internal uterine orifice than normal, and the thickness of the myometrium from the GS to the front of the uterus was 2.9 mm. When performing TVUS again after confirming a urine-filled bladder, the thickness of the myometrium from the GS to the bladder was 1.2 mm  parity and 6.4 ± 2.2 (5–40) days, respectively. In total, 184 patients were treated with a gonadotropin-releasing hormone analogue (GnRHa) before surgery. The overall average length of the operation time in the 323 patients was 206.8 ± 57.2 (94–504) min, and the average blood loss volume was 132.4 ± 196.8 (0–1500) ml. When concomitant PLA or LC was performed, the operation time was significantly prolonged, but the blood loss volume was not significantly different. Of the 323 patients, 143 patients had no complaints, but the other 280 patients had various symptoms, such as menstrual disorders and abdominal distension; one patient reported multiple symptoms. According to MRI, the average size of the dominant leiomyoma in 276 patients was 62.4 ± 29.2 (15–176) mm. Other notable findings besides subserosal or intramural leiomyomas were also detected, including submucous leiomyoma (n = 70), adenomyoma (n = 77) and ovarian tumour (n = 40). The average haemoglobin concentrations before the operation, immediately after the operation and before discharge were 13.0 ± 1.2 (6.6–16.7), 11.2 ± 1.3 (6.7–15.0) and 11.3 ± 1.3 (7.9–15.5), respectively. Autologous blood donation was performed in 230 patients, and blood transfusion was performed in 20 patients, including 19 patients who required autologous blood transfusion and one patient who required allogeneic blood transfusion. The annual number of TLH cases increased annually, and approximately 60% of the 323 TLH cases were performed in 2018 or 2019.As shown in Table We performed 323 TLHs for leiomyoma/adenomyoma (278 cases), low-grade (pre)malignant uterine tumour (40 cases), and other rare uterine diseases ; (2) “ovarian tumour” ; and (3) “heavy uterus” . “Gynaecological surgical history”  and “large leiomyoma”  showed a tendency to increase the possibility of intraoperative complications. However, no factor affected the rate of postoperative complications.To detect the significant factors affecting the possibility of intraoperative and postoperative complications, multivariate analysis of 11 representative factors was performed Table . Accordin = 13/17 vs. 64.1%, n = 196/306, P = 0.30). TLH also had another advantage related to diagnostics. After operations, we directly compared the imaging diagnosis according to MRI to the definitive diagnosis confirmed by pathological findings. These pathological data directly acquired through surgical procedures are valuable since hysterectomy for these indications was generally not selected as a first choice treatment [To manage the increasing indications for TLH, we must analyse the results of various cases and validate the safety of the procedure for these indications. In our hospital, from 2015 to 2019, approximately one-seventh of all TLHs were performed for indications other than leiomyoma/adenomyoma (45/323 cases). In these 45 cases, we treated 5 rare uterine cases without any serious complications; the rare cases included uterine haematoma, caesarean scar pregnancy, cervical ectopic pregnancy, vaginal atresia and cervical tumour formed by lymphatic and vessel dilation  as an intraoperative complication, but we could not consider the effect of operation time. When compared with published data from large hospitals in Japan , 12-20, This study identified risk factors for surgical complications of TLH, and other indications besides leiomyoma/adenomyoma did not increase the occurrence of complications. By considering these factors, the indications for TLH might be able to be expanded. With the increase in rare indications for TLH, a more accurate determination of the treatment approach can be achieved.


Streptococcus thermophilus, isolated as gently as possible, and commercial dextran were analyzed for molecular mass distributions as representative measure of molecule alterations. Generally, acid, excessive heat and ultrasonication, intensified by simultaneous application, showed EPS degradation effects. Thus, recommendations are given for isolation protocols. Ultrasonic degradation at 114 W/cm² fitted into the random chain scission model and followed third- (S. thermophilus EPS) or second-order kinetics (dextran). The degradation rate constant reflects the sensitivity to external stresses and was DGCC7710 EPS > DGCC7919 EPS > dextran > ST143 EPS. Due to their exceptional structural heterogeneity, the differences could not be linked to individual features. The resulting molecular mass showed good correlation (r² = 0.99) with dynamic viscosity.During isolation, exopolysaccharides (EPS) from lactic acid bacteria are subject of thermal, chemical, enzymatic or ultrasound stress of different intensity that may affect macromolecular properties, for instance molecular mass or (intrinsic) viscosity. These parameters are, however, crucial, as they are associated with the technofunctional potential of EPS replacing commercial thickeners in nonfermented products. The aim of this study was to systematically examine treatments EPS are usually exposed to during isolation and to investigate the underlying degradation mechanisms. Solutions (1.0 g/L) of EPS from  Streptococcus thermophilus, Lactococcus lactis or Lactobacillus delbrueckii ssp. bulgaricus are widely used as starter cultures. Associated to their growth, some strains of these species are able to produce exopolysaccharides (EPS) in situ and are thus beneficial for the texture and sensory properties of the fermented product, as EPS enhance the water binding capacity, reduce syneresis, increase the viscosity of milk gels or enhance their stability against shearing  is defined as the power per area of the sonicator probe tip A [cm²]:The energy input of the ultrasonic disintegrator was determined calorimetrically ,40. The In our experiments, a probe tip with a radius of 1.6 mm was used, and a linear correlation (r² = 0.99) was observed between the amplitude adjusted at the ultrasonic disintegrator and the power input (0.8 W ≤ P ≤ 9.1 W), resulting in an ultrasound density ranging from 10.0 to 114.2 W/cm².M [Da] and the number average molecular mass MN [Da]  and described by the weight average molecular mass Mils, see ). The poThe mono- and disaccharide content of EPS solutions was analyzed with HPLC-RI as described previously . DetectiThe dynamic viscosity η [mPa·s] of aqueous exopolysaccharide solutions (assumption: ρ = 1.0 g/mL) was determined with a LOVIS rolling ball viscometer  . The roln > 2) or arithmetic mean ± half deviation range (n = 2). Differences were statistically tested with the Student–Newman–Keuls test (p < 0.05) after performing one-way analysis of variance (ANOVA) using SAS® University Edition 6p.2 .Data are expressed as arithmetic mean ± standard deviation , and MM of dextran from L. mesenteroides was approx. one magnitude lower (2.29 × 105 Da). These MM were not significantly affected by thermal treatment at 60 or 80 °C for up to 60 min . This effect was overlaid by a heat-triggered molecule breakdown which was accelerated at higher temperature and longer incubation time and in accordance with findings from other authors: A decreased MM and lower apparent viscosity after a heat treatment was observed for scleroglucan  and MM,t [Da] refer to the weight average molecular mass of untreated EPS and EPS after a certain ultrasonication time t [min], respectively, and k′ [1/min], k′′ [1/(Da∙min)] and k′′′ [1/(Da2∙min)] to the rate constants for first-, second- and third-order degradation, respectively, under defined ultrasonication conditions . The effect of ultrasonication on a polymer is thus reflected by k: a lower k refers to increased resistance and therefore a lower decrease of MM.MThe weight average molecular mass of ultrasonicated EPS was fitted into the outlined functions . None ofM,0. Therefore, EPS with different initial molecular mass can be compared regarding their shearing resistance by means of the rate constant k′′′ at I = 114 W/cm². DGCC7710 fEPS were most vulnerable to ultrasonication, followed by DGCC7919 and dextran. ST143 fEPS showed the highest resistance against sonication as its k′′′ was lowest. With decreasing ultrasound density, k′′′ decreased, as indicated by the data for DGCC7710 fEPS. It still remains unclear whether the different behavior of dextran is due to the structural differences compared to EPS from S. thermophilus.Wu et al.  showed tOverall, our experiments showed that the EPS investigated in this study reacted differently to the thermal, chemical, enzymatic and ultrasound treatment. A higher sensitivity to chemical and thermal stress was in accordance with the observed sensitivity to mechanical energy input created by ultrasonication and was in the order DGCC7710 fEPS > DGCC7919 fEPS > dextran > ST143 fEPS. The EPS differ strongly in their chemical structures . Other tN,∞ was determined experimentally by sonication with an ultrasonic disintegrator for three hours as proposed earlier by Wu et al. [After linearization of Equations (1) and (2), the number average molecular mass of the EPS can be used to determine the localization of chain scission during ultrasonication. Mu et al. . Data ofM, evident for ultrasonication and shearing with a microfluidizer or a cell disruption system, indicates the same degradation mechanism of molecule breakdown for the different treatments. This is also consistent with findings from NMR experiments where we observed that microfluidization does not alter EPS structure [Leuconostoc citreum [Sargassum pallidum [L. mesenteroides [The linear dependency of η on Mtructure . Studiestructure ,44,50,51tructure , konjak tructure ,33, pect citreum  or polyspallidum  and six teroides , howeverM decrease was only 8% at maximum after 10 min at 90 °C, we suggest this step for cEPS detachment or resolvatization of isolated EPS. Furthermore, applying combinations of heat with other treatments simultaneously should be avoided, as this enhanced the degrading effect of ultrasound or pH . EPS degradation can be further reduced by replacing acid precipitation by enzymatic hydrolysis for protein removal. The ultrasonic disintegrator was shown to be inappropriate for EPS isolation because it rapidly reduced MM of all the exopolysaccharides. On the other hand, it may be useful for a targeted modification of EPS, especially at lower power density, and for the investigation of degradation mechanisms and kinetics. We observed that all EPS chains were broken randomly (Schmid model) following third-  or second-order kinetics (dextran). The sensitivity to ultrasonication at 114 W/cm² was highest for DGCC7710 fEPS (k′′′ = 1.80 × 10−11), followed by DGCC7919 fEPS (2.02 × 10−11), dextran (3.60 × 10−12) and ST143 fEPS (5.15 × 10−12). The data confirmed a linear relationship between MM and η, as recently published for other shearing systems. Ultrasonication showed to be a promising tool to easily adjust η and MM of EPS by selecting the appropriate sonication parameters  after empirical calibration. This allows to obtain EPS isolates with desired functionality for their use in nonfermented foods and beverages.The investigated EPS were largely affected by acid and ultrasonication treatment. As the M


The purpose of this meta-analysis was to comprehensively collect randomized controlled trials (RCTs) to assess the clinical efficacy of intrathecal morphine (ITM) versus local infiltration analgesia (LIA) in the treatment of total knee and hip arthroplasty patients.Relevant studies were identified from the Embase, PubMed, Cochrane Library, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure (CNKI) databases. We also reviewed the references of all identified articles to identify additional studies. For each study, we assessed the risk ratio (RR), weighted mean difference (WMD), and corresponding 95% confidence interval (95% CI) to synthesize outcomes. Meta-analysis was performed with Stata 12.0 software.P < .05). LIA significantly decreased cumulative morphine consumption by 13.52 mg. Moreover, the length of hospital stay was lower in the LIA group than in the ITM analgesia group. Finally, LIA significantly reduced morphine-related complications .We included 13 studies with 942 patients for meta-analysis. LIA significantly decreased the pain value with rest or mobilization until 72 hours (LIA was an effective approach for relieving postoperative pain and reducing postoperative consumption of morphine compared with ITM in total knee and hip arthroplasty patients. However, pain following TKA and THA in patients has been reported to be a common postoperative complication.Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are the most common procedures in orthopedic surgeries that can relieve joint pain and improve joint function. Moreover, effective pain control can quicken the recovery time and reduce the length of hospital stay.,6Careful pain management is necessary after TKA and THA to achieve early postoperative mobilization while ensuring patient comfort throughout. conducted a randomized controlled trial and revealed that LIA conferred superior analgesia to ITM following TKA in patients. Hess et al also suggested that ITM may not have a morphine-saving effect and may increase the occurrence of pruritus at doses over 0.3 mg. However, different opinions exist. Kaczocha et al indicated that ITM reduces postoperative pain in TKA patients.There are many analgesic options available for TKA and THA that vary in terms of efficacy and potential complications. Local infiltration analgesia (LIA) and intrathecal morphine (ITM) are 2 alternatives for pain management in TKA and THA patients. Some controversies remain, however, regarding the pain control efficacy and safety of these 2 methods. McCarthy et alTherefore, it was necessary to conduct a systematic review and meta-analysis to compare the efficacy and clinical outcome of LIA and ITM in TKA and THA patients. We hypothesized that LIA was superior to ITM for pain control without increasing adverse events in TKA and THA patients.2The Cochrane Handbook for Systematic Reviews of Interventions was followed during the performance of this meta-analysis, and the full texts were written in accordance with the PRISMA checklist . Because the meta-analysis was based on aggregate data, ethical approval was not required.2.1Two individuals independently reviewed the Embase, PubMed, Cochrane Library, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure (CNKI) databases for relevant articles published through March 2020. We also reviewed the references of all identified articles to identify additional studies. The search terms were as follows: “local infiltration anesthesia,” “anesthesia, local,” “infiltration anesthesia,” “anesthesia, infiltration,” “local anesthesia,” “intrathecal morphine,” “intraspinal injections”; “injections, intraspinal,” “injection, intraspinal,” “intraspinal injection,” “spinal injections,” “injection, spinal,” “injections, intrathecal,” “total knee arthroplasty,” “total knee replacement,” “TKA,” “TKR,” “total hip arthroplasty,” “total hip replacement,” “THA,” “THR,” “random, randomized controlled trial,” and “RCT.” These key words and mesh terms were combined with “AND” or “OR.”2.2The PRISMA guidelines were followed for the inclusion of studies in the meta-analysis. Following the PICOS  principle, the key search terms included (P) patients with total knee or hip arthroplasty; (I/C) patients treated by LIA or ITM; (O) the outcomes including the related clinical indexes ; and (S) RCT.The exclusion criteria included subjects in animal studies, cadaveric studies, in vitro studies, studies with a single cohort, case reports, and articles published in a form other than clinical trials. The article review procedure was conducted by 2 independent authors. If there was any disagreement between the 2 authors regarding the inclusion criteria of the related articles, a third author was consulted.2.3The data extraction was conducted by 2 independent authors separately by using a standardized form and recorded in Microsoft Excel . The following data were extracted: first author, publication year, Jadad score, intervention of LIA and ITM, sample size, age, female patients, and surgery type. The clinical outcomes included VAS with rest or mobilization until 72 hours, cumulative morphine consumption, length of hospital stay, occurrence of nausea and vomiting, pruritus, and respiration depression. Differences and disagreements were resolved by consensus. If the trials had more than 2 groups, we only extracted the interest-reported information and data. Pain intensity was assessed using the VAS pain score .2.4 A Jadad score of 1 to 2 was considered low quality, and a Jadad score of 3 to 5 was considered high quality.The quality of the included studies was assessed independently by 2 reviewers (Bao-chang Qi and Jing Yu) using the Jadad score, which evaluates studies based on randomization appropriateness, blinded outcome assessments, and complete descriptions of patients lost to follow-up.2.5I2: when I2 > 50%, suggesting significant heterogeneity, a random-effect model was used in the pooled result; in contrast, a fixed-effect model was employed when I2 < 50%. Heterogeneity among the included studies was analyzed by the Chi-square test and I2. The Z test was used to evaluate the overall effect.Meta-analysis was performed with the support of Stata 12.0 software . For dichotomous outcomes , the results were calculated with risk ratios (RRs) and 95% confidence intervals (95% CIs). For continuous outcomes with normal distributions, weighted mean differences (WMDs) and 95% CIs were used to test the overall effects. Random- or fixed-effect models were adopted depending on the value of 33.1,11–22 for this meta-analysis, incorporating 942 patients. The study selection is outlined in Fig. We reviewed a total of 504 articles identified by our initial keyword search, of which 119 were excluded following de-duplication by EndNote X7 (Thomson Reuters). Then, 342 studies were excluded after screening the title and/or abstract. After full-text reading and screening, the remaining 13 articles were retrieved and read, and no studies were excluded according to the inclusion criteria. Finally, we included a total of 13 studies3.2Table 3.3P = .000), 12 hours , 24 hours , 48 hours , and 72 hours .The summary results of LIA versus ITM in terms of the VAS scores with rest up to 72 hours are summarized in Table 3.4P = .000; Table P = .000; Table P = .198; Table P = .012; Table P = .000; Table The combined results showed a significantly lower pain score with mobilization in the LIA group at 6 hours  provided data on the total morphine consumption. The results showed that there was statistical heterogeneity in the total morphine consumption  reported outcomes related to the length of hospital stay. There was high heterogeneity between the included studies  reported data on the occurrence of nausea and vomiting. There was slight heterogeneity between the included studies  were eligible for inclusion in the meta-analysis for the frequency of pruritus. Heterogeneity between the included studies was absent  mg. We concluded that the above results were similar to the results of our meta-analysis. We found that LIA significantly reduced morphine consumption by approximately 13.52 mg (95% CI: −19.09 to −7.95).We next evaluated morphine consumption. Morphine consumption was reduced following TKA and THA when patients received LIA rather than ITM. Askar et alIn economic terms, LIA provides a shorter hospitalization time and, last but not least, a better cosmetic outcome than ITM. LIA can also contribute to postoperative pain control as previously documented as a premise for patients’ fast recovery in order to reduce bed-related complications. Moreover, surgeons should pay more attention to morphine-related complications. LIA can significantly reduce morphine-related complications  compared with ITM.However, this study has the following limitations: the low number of subjects studied and the inadequate sample size within studies; the variable dose of LIA and ITM between studies; the variability of the rehabilitation program between studies, thus potentially affecting the length of hospital stay; and most significantly, the heterogeneity contributing to different doses and intervals among the included studies.5On the basis of the results of the present analysis, the LIA group had more advantages than ITM in terms of VAS scores during early postoperative periods; moreover, the LIA group may have been subject to lower morphine consumption. Furthermore, participants under LIA did not suffer worse results regarding postoperative complications and VAS scores at 3 months postoperatively. However, further studies with standardized, unbiased methods, and larger sample sizes are still required for deeper analysis.Conceptualization: Wei-song Qiao.Methodology: Wei-song Qiao.Software: Bao-chang Qi.Writing – original draft: Bao-chang Qi, Jing Yu.Writing – review & editing: Jing Yu.


Under these “pathological” conditions, dermal–epidermal split formation and dyskeratosis were prominent in organ-cultured human skin, and epidermal reepithelialisation was significantly impaired (p < 0.001), associated with reduced keratinocyte proliferation (p < 0.001), cytokeratin 6 expression (p < 0.001) and increased apoptosis (p < 0.001). Moreover, markers of intracutaneous angiogenesis (CD31 immunoreactivity and the number of of CD31 positive cells and CD31 positive vessel lumina) were significantly reduced. Since we had previously shown that thyroxine promotes wound healing in healthy human skin ex vivo, we tested whether this in principle also occurs under “pathological” wound healing conditions. Indeed, thyroxine administration sufficed to rescue re-epithelialisation (p < 0.001) and promoted both epidermal keratinocyte proliferation (p < 0.01) and angiogenesis in terms of CD31 immunoreactivity and CD31 positive cells under “pathological” conditions (p < 0.001) ex vivo. This demonstrates the utility of this pragmatic short-term ex vivo model, which recapitulates some key parameters of impaired human skin wound healing, for the preclinical identification of promising wound healing promoters.Impaired cutaneous wound healing remains a major healthcare challenge. The enormity of this challenge is compounded by the lack of preclinical human skin wound healing models that recapitulate selected key factors underlying impaired healing, namely hypoxia/poor tissue perfusion, oxidative damage, defective innervation, and hyperglycaemia. Since organ-cultured human skin already represents a denervated and impaired perfusion state, we sought to further mimic “pathological” wound healing conditions by culturing experimentally wounded, healthy full-thickness frontotemporal skin from three healthy female subjects for three days in either serum-free supplemented Williams’ E medium or in unsupplemented medium under “pathological” conditions (i.e. hypoxia [5% OThe online version of this article (10.1007/s00403-020-02092-z) contains supplementary material, which is available to authorized users. Chronic skin ulceration, particularly in the context of an ageing population, is a complex challenge in daily medical practice and is associated with (i) significant morbidity, (ii) impaired quality of life and (iii) substantial health care costs . Indeed,Effective wound healing (WH) is a metabolically demanding process that relies upon the co-ordinated action of multiple tightly regulated complex biological processes, such as the timely initiation and termination of inflammation, adequate cell proliferation and efficient tissue remodelling, all of which are dependent upon sufficient tissue oxygenation, energy supply and innervation , 22, 62.Despite the presence of a multitude of both in vivo , 61, 69 Furthermore, the available human WH models typically lack most resident skin cell populations and skin appendages, for example, the hair follicle (HF) , 16 and,However, even these models often fail to recapitulate the factors complicating WH in vivo, namely hypoxia, oxidative stress and hyperglycaemia. Therefore, based on previously published full-thickness human skin WH ex vivo models , 74, 75,In the second part of our study, we sought to ascertain the extent to which an agent known to promote WH in the human full-thickness skin ex vivo WH model, namely thyroxine (T4), also exerts WH-promoting effects in the “pathological” WH model. For this, we chose to test T4 as the lead compound, because it has been shown to promote cutaneous WH both in mice in vivo , 54 and Temporal and occipital region scalp skin samples from six females (aged 52–65 years) were obtained as by-products from cosmetic surgical procedures. All patients provided informed consent, and the study was approved by the Institutional Research Ethics Committee at the University of Luebeck . All experiments were performed in accordance with the ethical standards set by the Declaration of Helsinki and its later amendments.Skin samples from the first three patients were used to establish a “pathological” WH model and skin samples from the remaining subjects were used to test the effects of T4, under standard and “pathological” conditions (see below), on several WH-related parameters. Skin fragments were “wounded” via a 4-mm punch biopsy, with the tissue periphery, which rapidly shows signs of epiboly, i.e. the tendency of the epithelium to rapidly enclose traumatically exposed mesenchyme, , 58 takeThe punch biopsies were then placed in standard supplemented Williams’ E medium (WE) (22), or in the modified “pathological” medium. All punches were cultured in a six-well plate, each well containing 3 ml of medium and two skin punches, floating at the liquid–air interface. After completion of the organ culture, skin punches were snap frozen in cryomatrix and liquid nitrogen and stored at − 80 °C until cryosections were cut, stained and analysed. See Supplementary Fig. Serum-free WE supplemented with l-glutamine (2 mmol/l), hydrocortisone (10 ng/ml), insulin (10 μg/ml) and streptomycin (10 µg/ml)/penicillin G (100 IU/ml) served as the standard control culture medium , 37, 41.The “pathological” WH conditions included hyperglycaemia, achieved by the addition of glucose. Given that diabetes can be diagnosed with a random serum glucose concentration of ≥ 11.1 mmol/dl (≥ 200 mg/dl), and that tissue damage due to hyperglycaemia is a long-term complication , 55, we Hyperglycaemia results in the formation of advanced glycation end-products (AGEs). The formation of AGEs is also promoted by oxidative stress and leads to tissue dysfunction by the cross-linking of collagens, activation of macrophages and covalent bonding of lipoproteins . Under “2O2) per well (concentration 10 mM) based on our prior experience with experimental induction of oxidative damage in human skin appendages [Oxidative damage was experimentally induced by adding 3.06 μl of hydrogen peroxide  . DyskeraEvaluation was performed with Image J software by measuring the length and area of the ET. Photomicrographs were obtained using the Keyence-Biozero 8000. Dimensions were orientated via the insertion of a 50 µm scale bar. Immunofluorescence (IF) microscopy for the employed read-out parameters  was performed and analysed as described previously , 41.Proliferation and apoptosis were determined by Ki-67/TUNEL double-IF staining. The total number of Ki-67, TUNEL and DAPI was counted in a defined area inside the epidermis and ETs. In the first experimental set-up, Ki-67 positive and TUNEL positive  cells were counted inside a defined area of the ET but also in three additionally defined areas of epidermis. In the second experimental set-up (i.e. addition of T4), evaluation of apoptosis and proliferation was restricted to the ETs.CK6 expression was measured given that it is increased in both hyperproliferative and wounded epithelium , 70 and Angiogenesis, another key WH parameter, was assessed by the detection of CD31 positive cells, a validated endothelial cell marker , 74. Thrt-test was used when comparing two groups. When the data were not normally distributed, the Mann–Whitney U test was selected. Statistical analyses were also performed using Graph Pad Prism v. 3.00 . All data were expressed as mean ± SEM (standard error of the mean); p values * < 0.05, ** ≤ 0.01, *** ≤ 0.001 were regarded significant. For each parameter, two punches were cultured and compared. All data were pooled, resulting in a total of 19–54 skin fragments per experimental group, derived from three different female patients to establish the model and from three female patients to test the effect of thyroxine. Data pooling was justified since the obtained results in at least three independently run experiments showed comparable trends.All data sets were tested for normal distribution using Graph Pad Prism v. 3.00 . When the data set was normally distributed, the First, we tested the working hypothesis that “pathological” WH conditions would negatively effect skin structure and integrity as well as re-epithelialisation, even in short-term organ culture. After wounding, KCs migrate from the BL along the wound edge to restore the epithelial barrier after wounding,  which caMoreover, the number of intraepidermal dyskeratotic cells and the extent of the epidermal separation from the BL(epidermolysis) were both significantly increased in the skin under “pathological” WH conditions compared to standard human skin organ culture conditions Fig. c–f. ThisNext, we examined how “pathological” WH conditions influenced epidermal KC apoptosis and proliferation ex vivo. KC apoptosis was significantly increased throughout the entire epidermis under “pathological” conditions compared to that in control conditions Fig. a, b. MorGiven that CK6 protein expression is strongly upregulated in wounded human epidermis and during epidermal regeneration  and thatTo complete the characterisation of our “pathological” WH model, we asked whether hyperglycaemia, hypoinsulinaemia, hypoxia and oxidative stress also impact upon angiogenesis since we had had previously shown that indications of angiogenesis are still visible, albeit a fluctuating levels, even after several days of organ-culturing human skin under serum-free conditions , 74. MarIn summary, these findings demonstrate that the simple, easily set up organ culture system for experimentally wounded skin reported here rapidly generates severely healing-impaired previously healthy human skin within just three days ex vivo. Dyskeratosis and split formation were measured to confirm that the skin was being sufficiently stressed by the “pathological” conditions. Having confirmed this, we next addressed the ability of T4 to influence the key parameters of WH, namely re-epithelialisation and neo-angiogenesis under “pathological” conditions.Most WH assays employed in vitro, ex vivo, or in vivo examine the impact of test agents and experimental manipulations under WH conditions that reflect acute WH in essentially healthy tissue. Unsurprisingly, WH-promoting effects seen under these more or less physiological conditions often do not translate into tangible clinical benefits under the only WH conditions that really matter in clinical practice: impaired WH and ulcer formation . TherefoWe, therefore, explored whether or not the candidate WH-promoting thyroid hormone, T4 , 74, posp < 0.01) and under “pathological” (0.001) conditions  culture conditions ex vivo. Moreover, the inhibitory effects of these culture conditions on ET formation  were reversed by T4 supplementation (Supp. 1). In addition, T4 tended to restore the reduced CK6 expression in the ET under “pathological” WH conditions  in particular. Therefore, we sought to establish a robust and clinically relevant “pathological” WH model and determine the extent to which candidate WH-promoting agents could modulate WH under these carefully defined and reproducible conditions.2O2 and reactive oxygen species (ROS), resulting from reduced perfusion in peripheral vascular disease, also disrupts WH in vivo [For example, hyperglycaemia and hypoinsulinaemia or insulin end-organ resistance are known to impair WH via a reduction in dermal fibroblast proliferation and migration. Furthermore, the low-grade inflammation and angiogenesis associated with diabetes and the metabolic syndrome, respectively, also contribute to impaired WH and tissue remodelling . In addi in vivo .Here we have developed and validated a pragmatic, well-defined, serum-free human skin organ culture model, in which the epidermal repair and angiogenesis can be studied under severely healing impaired  WH conditions that mimic ex vivo, at least to some extent, the hypoxia, oxidative damage, impaired perfusion and innervation, hyperglycaemia and hypoinsulinaemia which may be present in diabetic ulcers in vivo. This model recreates impaired WH-associated alterations in KC morphology, reduced KC proliferation and migration, increased KC apoptosis, epidermolysis, and reduced angiogenesis and is, therefore, well suited for the preclinical testing of candidate WH-promoting agents that hope to positively affect these parameters. That T4 was able to partially “rescue” epidermal repair and promote angiogenesis, even under these severely healing-impaired clinically relevant WH conditions, in a human model, is interesting and potentially clinically relevant..With the aim of validating our “pathological” WH model of human skin, we selected T4 as a candidate WH promotor based on its effects on mitochondrial biogenesis, KC proliferation and angiogenesis , 68. TheThe pro-angiogenic effect of T4 is mediated by the cell surface receptor integrin αvβ3. Stimulation of this receptor on endothelial cells stimulates intracellular transcription of pro-angiogenic growth factors (GFs), like bFGF and VEGF .Induced hypothyroidism in mice results in papillary rarefaction and could be treated via systemically applied tri-iodothyronine (T3) which induced sprouting angiogenesis in the heart tissue of the hypothyroid mice . There iSpecifically, in terms of WH, T4 has been shown to have a WH-promoting effect compared to untreated groups in animal studies , 54. T3 In terms of dissecting the molecular mechanism underlying the effect of T4 on WH in this model, future studies may wish to examine the extent to which fibroblast and HF stem cell proliferation contribute to the WH evidence in the model, for example, by determining matrix metalloproteinase, heat-shock protein and CK 15 expression, respectively. Indeed, therapies aimed at mobilising stem cells have recently be shown to promote cutaneous WH in diabetic rats . MoreoveIn summary, we report the development of a pragmatic organ culture assay that quickly transforms viable biopsy-wounded human skin into a “pathological” state, where WH is severely impaired. We have attempted to provide proof-of-principle that (i) human skin with severely “pathological” WH features is still capable of responding in a therapeutically desired manner to treatment with a candidate WH promoter and (ii) TH-R agonists are interesting, but as yet insufficiently investigated candidate promoters of “pathological” WH. This novel preclinical test system, which is characterised by severe hypoxia, excessive ROS levels, hyperglycaemia and insulin withdrawal, can serve as a surrogate assay for probing the efficiency of candidate WH promoters under well-standardized and clinically relevant ex vivo conditions.Fig. 1 The demonstration of the experimental set-up. Full thickness human frontotemporal skin fragments were cultured in different medium conditions of clinically physiological (= standard) and “pathological” conditions treated or untreated by thyroxine (T4). (A) The 4 mm punch biopsy obtained from skin fragments after cosmetic surgery. Ki-67 stained images show the location of analysed areas inside the punch. (B) The composition of the “pathological” medium, Williams’ E. medium serves as a basic medium and was modified to induce the specific culture condition. (C) The timetable of the experimental setup. Fig. 2 Human organ cultured skin influenced by “pathological” factors reveal major differences in epidermal/dermal structure and T4 likely has a positive influence on keratinocyte migration. (A) Split formation (SF) (B) Dyskeratosis (C) length and (D) Area of the epithelial tongues (ETs). SF analysed by Periodic acid-Schiff staining was categorised into 4 stadiums: (Stage 4) > ¾ of the epidermis is detached, (Stage 3) > ½ of the epidermis is detached, (Stage 2) < ½ of the epidermis is detached and (Stage 1) < ¼ th of the epidermis is detached. Data of patient 2 and 3 show a very similar degree of SF in the “pathological” medium . Patient 1 also showed a higher but non-significant difference between standard and “pathological” medium. Dyskeratosis was concise under “pathological” culture conditions. Haematoxylin and Eosin stained sections of standard and “pathological” conditions were evaluated. Measurement of the ETs by Image J were standardised . The length and area of the ET varied dependent on the culture medium and whether T4 was added or not. Dyskeratosis, SF and ETs were analysed per visual field along the epidermis, detached and attached epidermis. Data are mean ±SEM of 3 patients. Significance relative to control data (standard medium) at the same time point denoted by *p<0.05 , **p<0.01, ***p<0.001, Mann-Whitney U test, t-test. Fig. 3 Primary and secondary antibodies. (PPTX 3475 kb)Supplementary file1 Below is the link to the electronic supplementary material.


Previous work has shown that when arrays of objects are grouped within clusters, participants can enumerate their numerosity more rapidly than when objects are randomly scattered, a phenomenon termed “groupitizing”. Importantly, the magnitude of the grouping advantage correlates with math abilities in children. Here we show that sensory precision of numerosity estimation is also improved when grouping cues are available, by up to 20%. The grouping can be induced by color and/or spatial proximity, and occurs in temporal sequences as well as spatial arrays. The improvement is strongest for participants with the highest thresholds in the random, ungrouped conditions. Taken together with previous research, our data suggest that measurements correlations between numerosity estimation and formal math skills may be driven by grouping strategies, which require a minimal level of basic arithmetic. Approximate Number System (ANS) and, like all sensory systems, is error-prone, with errors increasing proportionally with numerosity, obeying Weber Law4. Importantly, many studies have found a correlation between ANS precision (measured by Weber fraction or Coefficient of variation) and child math abilities, with lower precision associated with poorer performance in math8. Children with dyscalculia, a neurodevelopmental disorder affecting mathematical and numerical learning, often show higher Weber fractions compared to typically developing children10. Based on this evidence it has been proposed that the ANS may constitute a foundational non-symbolic system on which the language-based mathematical system could subsequently be built6.When counting is not possible, humans and other animals can rapidly estimate the number of items in any scene to achieve an approximate assessment of their numerosity. The system sustaining this ability is often termed the 11 first reported that estimates of numerosities less than four are fast and error-free, subsequently termed subitizing by Kaufman and Lord12. Subitizing is robust, and occurs for both sequential and simultaneous stimuli, in all sensory modalities16. Subitizing is highly dependent on attention24, and seems to work in parallel with the estimation system, boosting performance at low numerosities.Jevons25 suggested that subitizing mechanisms may also come into play for higher numerosities, a process they term "groupitizing". This is very much like George Miller’s well-known notion of “chunking”, where complex sets of information such as long telephone numbers can be more easily recalled if parsed into three or four smaller “chunks”. Starkey et al.25 measured counting speed of spatially clustered arrays in school-age children, and found that clustering, or grouping, increased performance. Crucially, both the number of clusters and the number of elements within each cluster was limited to the subitizing range (e.g. 7 = 2 + 2 + 3). Interestingly, the grouping advantage increased with age and correlated with arithmetic abilities, with more math-skilled children showing stronger groupitizing effects. More recently, it has been reported that grouping by color can also decrease reaction times in adults26. Overall these studies suggest that serial counting without time constraints may be not a "pure" and direct measure of ANS precision, but could be tempered by arithmetical strategies, such as grouping, which involves processes such as parse-and-add.More recently, Starkey and McCandlissIn this study, we ask whether grouping items by spatial proximity or color not only increases enumeration speed but also increases precision (measured as Coefficient of variation). We also investigated whether this grouping phenomenon is a general property of numerosity perception, applying to temporal sequences as well as spatial arrays. The results suggest that groupitizing occurs for estimation of both temporal and spatial dimensions of numerosity. We also observed a robust inter-individual variability in the magnitude of grouping-based improvement, with participants who were less precise in estimating numerosity in random arrays benefiting more from the groupitizing. This suggests that some participants may take advantage of intrinsic grouping in random arrays to increase their performance, therefore benefit less from the explicit experimentally induced grouping.Sixteen young adults  participated in this study . All participants had normal or corrected-to-normal vision. All completed all tasks except one, who was unavailable to perform the two sequential numerosity conditions.Comitato Etico Pediatrico Regionale—Azienda Ospedaliero-Universitaria Meyer—Firenze FI). The research was performed in accordance with Declaration of Helsinki and informed consent was obtained from all participants prior to each experiment.Stimuli were created with Psychophysics toolbox for Matlab and displayed on a 60 Hz—15″ screen monitor (MacBook Pro) placed at viewing distance of 57 cm. Subjects were tested in a quite, dimly light room. The experimental procedures were approved by the local ethic committee , then entered the response on the numeric keypad, which initiated the following trial. Response time was measured from the stimulus offset to the beginning of vocalization. Participants were asked to respond as soon as possible, but also to concentrate on accuracy. Each condition was tested in separate blocks, and participants were never explicitly informed about the grouping cues.Numerosity levels ranged from 4 to 16 . In the structured conditions, each numerosity was organized into clusters (between 2 and 4), each containing a variable number items (between 2 and 6), resulting in the following configurations: 2, 2−2, 2, 1−3, 3−2, 2, 2−3, 3, 1−3, 3, 2−2, 2, 2, 2−4, 4−4, 3, 2−4, 4, 1−3, 3, 3−3, 3, 3, 1−4, 4, 2, 1−3, 3, 3, 2−3, 3, 3, 3−4, 4, 4−4, 4, 3, 3−4, 4, 4, 3−5, 5, 3−4, 4, 4, 4−5, 5, 6. As numerosities 4 and 16 were not analyzed , each grouped pattern comprised a minimum of 2 and a maximum of 4 clusters. All clusters except one  contained from 1 to 4 elements. On each trial, a given numerosity and configuration pattern were randomly selected. Each participant completed about 150 trials for each of the six conditions .Stimuli were arrays of squares (0.4° × 0.4°) displayed for 500 ms on each trial. Squares could not overlap and were constrained to fall within a 12°X12° virtual square area. In the conditions where spatial structure was manipulated, the individual items were white squares within black borders (so luminance was not a cue to number). In the unstructured conditions, the position of each square was randomly selected from 169 possible positions (within the stimulus area), being the centers of equally spread sectors within the 12°X12° area (each grid 1°X1°). For the spatially grouped condition, stimuli were arranged in 4 possible groups of 12 possible positions . Color was assigned from left to right, so that similar colors appeared in vertical rows. For example, in the 3, 3, 2 condition depicted in Fig. Stimuli were streams of 3° × 3° squares each presented at screen center for 70 ms, for a total trial duration of 3 s Fig. D. The enSince participants were explicitly informed about the numerical range (4–16), we eliminated the two extreme numerosities from the analyses. We controlled for response outliers by eliminating trials with RTs longer than 3 standard deviations from the average response time, calculated separately for each numerosity level and participant.i. Improvement (I) by grouping was measured by a normalized index yielding the proportion improvement:For each participant, we calculated for each numerosity the average perceived numerosity, the standard deviation of the responses and the median response time. Precision was measured by normalizing the standard deviation by the physical numerosity yielding the Coefficient of variation (CV), a dimensionless index of precision that allows comparison and averaging of performance across numerosities.2, using JASP and Matlab.Data were analyzed by repeated measures ANOVAs, and effect sizes were reported as η25 with both the number of groups and the number of items/events within each group falling within the subitizing range: 2, 3 or 4 groups each containing 1, 2, 3 or 4 items/events.We asked participants to estimate the numerosity of briefly presented visual impulses, presented either in simultaneous spatial arrays or temporal sequences. For both conditions (tested in separate sessions), we investigated the effects of task-irrelevant grouping cues on numerosity estimation precision and speed. Grouping manipulations mainly followed the formal definition of Starkey and McCandliss = 834.289, p < 0.001, η2 = 0.982; sequential: F = 282.289, p < 0.001, η2 = 0.953), but there was no significant effect of “condition”  = 1.285, p = 0.29, η2 = 0.08; sequential: F = 0.281, p = 0.60, η2 = 0.02) and the condition-by-numerosity interactions were insignificant  = 0.742, p = 0.84, η2 = 0.047; sequential: F = 0.311, p = 0.97, η2 = 0.022). Overall, these results clearly indicate that grouping did not significantly affect average perceived numerosity.We first evaluated the effect of grouping on the accuracy of estimation of perceived numerosity. Figure Having established that grouping did not change average perceived numerosity (accuracy), we investigated its effect on sensory precision, indexed by Coefficient of variation Eq. . This is = 4.9, p = 0.005, η2 = 0.247), with grouping decreasing Coefficient of variation compared to the spatially random condition  = 4.921, p < 0.001, η2 = 0.634), suggesting that Coefficient of variations are not constant with numerosity, while the interaction was not  = 1.365, p = 0.097, η2 = 0.08), suggesting that the overall effect of grouping was constant across numerosity levels.For spatial presentations, Coefficient of variation was highest for the non-grouped condition, higher than all the grouped conditions. Repeated measures ANOVA with numerosity (11 levels) and condition (4 levels) revealed a significant main effect of condition  = 9.38, p = 0.008) and by color gradient in space  = 13.908, p = 0.002) both induced a significant reduction of Coefficient of variation, and both had a quite large effect . Grouping by color without spatial gradient did not produce a significant reduction in Coefficient of variation  = 2.264, p = 0.15, η2 = 0.13). The ANOVA comparing the two color conditions  revealed that grouping by color with a gradient in space produced a significant reduction in Coefficient of variation compared to color alone  = 5.165, p = 0.038, η2 = 0.256). The interaction between numerosity-by-condition was never significant (p > 0.05) in any condition comparison  = 11.683, p = 0.004, η2 = 0.455). Once again, the numerosity-by-condition interaction was not significant (p > 0.05).Figure 26, we also investigated the effect of grouping in term of response speed  = 1.008, p = 0.40, η2 = 0.06). However, separate repeated measure ANOVAs against spatially random stimuli revealed that grouping by spatial structure significantly reduced RTs from 2.02 ± 0.26 to 1.86 ± 0.22 s, an effect of 8%  = 4.612, p = 0.048, η2 = 0.235, for all the other ANOVAs min p = 0.25). There was a significant reduction of response time induced by grouping of temporal sequences  = 8.861, p = 0.01, η2 = 0.388). Again, the effect of numerosity was statistically significant  = 10.13, p < 0.001, η2 = 0.42) but not the numerosity-by-condition interaction  = 0.924, p = 0.513, η2 = 0.062). Finally, all ANOVAs revealed a statistically significant effect of numerosity , but no numerosity-by-condition interactions (p > 0.05).Repeated measure ANOVA with numerosity (11 levels) and condition (4 levels) as factors did not reveal a significant effect of spatial grouping condition  and temporal color clustering—the correlation was significantly positive . On the other hand, the condition in which grouping did not yield a significant advantage on numerosity precision (random space Vs random color in space), showed no significant advantage (p = 0.10).This study shows that using color, or spatial or temporal proximity to group items together robustly improves the precision of numerosity estimation, by up to 20%.28. It would be interesting to test much higher numerosities and densities, to see if grouping can also aid in judgments of texture density. It would seem unlikely if based on subitizing, as subitizing is limited to about 4, but worth verifying.The magnitude of the advantage for grouping did not vary with numerosity, over the range tested, from 5 to 15 Fig. . That is26. We found that grouping by spatial structure slightly reduced reaction times relative to the spatial random condition, by about 8%. However, RTs in the spatial gradient color condition were not significantly different from the random color condition. One possibility for this discrepancy may be that grouping by the spatial dimension is more salient compared with grouping by color. Alternatively, colored items may induce a strong tendency to automatically group the stimuli, even when randomly scattered spatially. This idea is supported by the lower CVs in the random condition with coloured stimuli compared to those measured with achromatic stimuli. Not surprisingly, this statistically insignificant trend was not evident in the RTs, in line with the fact that in the present study RTs have proven to be less robust in detecting grouping effects than CVs.We also found smaller and less robust advantages in reaction times, confirming previous studiesgroupitizing, speeds up serial counting25, but this does not help preschoolers. Furthermore, the grouping advantage correlated positively with arithmetical abilities in school-age children, suggesting that grouping relies, at least to some extent, on formal arithmetical knowledge. Thus grouping may reflect an implicit math strategy of numerosity perception, like “parse the scene into subitizable groups then sum the subitized estimates”. That grouping not only speeds counting but also lowers numerosity estimation thresholds has broad implications. Precision in numerosity estimation and discrimination are predictive of child math abilities29, and are both impaired in dyscalculia10. These results have been interpreted as a link between the perceptual ability to estimate numerosity and the cognitive ability to learn math15. However, if grouping strategies are spontaneously used by some participants, such as those with more spontaneous arithmetical skills, it could be this that mediates the link between numerosity and math proficiency. Use of grouping information, either intrinsic or explicitly introduced, requires some basic math skills, such as rapid addition of the numerosities of the sub-groups. It is likely that participants who opt for this strategy—rather than a global appraisal of the whole pattern—would be those with the greater math skills. This would have important implications for understanding the link between measures of numerosity sensitivity and math.Previous research has shown that grouping, or In the present study, participants who were more precise in the random condition benefited proportionately less from grouped configurations than those with higher thresholds. One plausible explanation for this is that those with lower thresholds use grouping strategies even with the random patterns, taking advantage of intrinsic grouping in randomness. These people may benefit less from the explicit grouping imposed by spatial or temporal proximity, and therefore show less improvement. This possibility is interesting, with implications about different individual styles in numerosity perception, well worth pursuing further.8, numerosity discriminations at high densities7 do not; nor does subitizing13. Furthermore, thresholds for temporal sequences do not predict math performance29, despite the clear evidence for a generalized number system encompassing space and time31. All this suggests that some aspect of estimation of numerosity at low densities is related to math. A clear candidate mechanism could be “groupitizing”, the use of strategic grouping to parse arrays into subitizable chunks. As mentioned above, this strategy requires some basic arithmetical skills: simple but rapid addition. It is reasonable to suppose that this skill does not help in the subitizing range, where arrays are already subitizable without further parsing, so that is not predictive of math. Similarly, for high numerosities the parsing strategy would not be effective, as only a limited number of subitizable sub-sets can be counted. Why estimation of temporal numerosity sequences does not correlate with math is less clear, as the present results show that a grouping strategy is possible with temporal sequences, and that those who benefit most from the grouping cues are those with highest thresholds. Perhaps the fact that temporal sequences are necessarily one-dimensional makes it harder to spontaneously group into sub-sets, particularly for young children. Also, in previous studies the presentations were constrained to be quite rhythmic, which does not lend to spontaneous parsing into groups. And perhaps phenomena such as “entrainment”33 tend to make the sequences even more rhythmical, and hard to group. Again, this idea bears further investigation, particularly with children.The correlation between numerosity precision and math skills is interesting. While thresholds for estimating numerosities at moderate, uncrowded densities predict well math performanceTo conclude, the current study demonstrated that use of grouping strategies can aid considerably in the estimation of numerosity. The strategy may be related to mathematical abilities, and understanding it better could be of considerable importance in understanding the link between estimating numerosity and formal math skills.


Multivariable linear regression analysis was performed to evaluate associations with left ventricular structural changes, corrected for body mass index (BMI) z score. Forty-three patients entered analysis, of whom 24 (55.8%) showed LVH. The most common geometrical change was eccentric LVH (eLVH), occurring in 21 subjects (48.8%). Demographic and anthropometric variables did not differ between patients with and without LVH. Independent of BMI z score, left ventricular mass index was significantly associated with apnea–hypopnea index (AHI) . Interventricular septum thickness (IVST) was significantly associated with HOMA-IR values , HDL-cholesterol , and triglyceride levels . LVH, especially eLVH, is highly prevalent amongst severely obese adolescents. Adverse changes in cardiac structure, increased IVST in particular, are independently associated with several nonhemodynamic comorbidities that are common in this population, namely OSA, insulin resistance, and dyslipidemia.Left ventricular hypertrophy (LVH) is independently associated with a higher risk of cardiovascular morbidity and mortality in adults. Adiposity is a risk factor for LVH, independent of blood pressure. Potential causes of this nonhemodynamic pathogenesis identified in adults include adverse body fat distribution, insulin resistance, dyslipidemia, and obstructive sleep apnea syndrome (OSA). In severely obese adolescents, the determinants of obesity-induced changes in left ventricular structure are poorly characterized. Cardiac ultrasonographic, demographic, anthropometric, and comorbidity-related data were prospectively collected in adolescents with severe obesity refractory to conservative treatment who presented for surgical therapy. Differences between adolescents with LVH and without LVH were evaluated using independent samples  Pediatric obesity is associated with adverse changes in left ventricular geometry, especially left ventricular hypertrophy (LVH) –6. In adThe reported prevalence of LVH in obese children, adolescents, and young adults between the ages of 3 and 22 years old ranges from 10 to 46% , 10. IntAdiposity is a predictor of LVH in both hypertensive and nonhypertensive children, independent of age –15. The The aim of our research is to improve understanding of the prevalence and determinants of left ventricular geometrical changes in a homogeneous and well-characterized population of severely obese adolescents. We hypothesize that LVH, especially eccentric LVH, is highly prevalent in our population, that BMI is the most important anthropometric predictor of LVH, and that LVH is independently associated with several pathogenic factors, including blood pressure and variables related to insulin sensitivity, lipid profile, hormonal aberrancies, and OSA.This study was conducted as part of a randomized controlled trial for adolescents eligible for bariatric surgery  . The stu2 (or ≥ 35 kg/m2 combined with presence of obesity-associated comorbidity); and participation in combined lifestyle interventions during at least 12 months without adequate weight loss . In order to maintain a homogenous study population with regard to pubertal status, girls were excluded if they were premenarchal, boys if their bone age was < 15 years.All patients included in the BASIC trial who received a baseline cardiac ultrasound examination were used for this baseline analysis. Detailed information regarding the BASIC trial study design, in- and exclusion criteria, and randomization process was published previously . In summAll participants were subjected to standardized comprehensive baseline measurements and investigations in order to exclude  conditions causing obesity.2, and BMI z scores were calculated using Cole’s LMS method /[body height*body height] in kg/mS method . Daytimee report .A fasting blood draw was performed to measure serum glucose, insulin, cholesterol, triglycerides, free fatty acids, glycated hemoglobin (HbA1C), and leptin. HOMA-IR was calculated according to the method described by Mathews et al., i.e., ((fasting insulin (microU/l)/(fasting glucose (mmol/l))/22.5, where insulin was converted from pmol/l to microU/l by dividing by 6.945 .Video-assisted 12 channel polysomnography (PSG)  was performed at the pediatrics department of Maastricht University Medical Center. The scoring of sleep stages and respiratory-related events were performed by a single specialized analyst using the American Academy of Sleep Medicine (AASM) 2012 updated guidance for scoring pediatric respiratory events .All cardiac ultrasounds were planned with a single pediatric cardiologist, who reported interventricular septal thickness (IVST), left ventricular end-diastolic posterior wall thickness (LVPWT), left ventricular end-diastolic dimension (LVEDD), and left ventricular end-systolic dimension (LVESD). In the absence of this pediatric cardiologist, another experienced pediatric cardiologist was consulted to perform the ultrasound examination.3 − LVEDD3)) + 0.6 [2.7 (height in meters). LVH was defined as a LVMI ≥ 38.6 g/m2.7, in accordance with the 95th percentile of LVM in a cohort of 192 healthy children of 6 to 17 years old [Left ventricular mass (LVM) was calculated using the Devereux formula, where left ventricular mass (in grams) is equal to 0.8(1.04((LVEDD + IVST + LPWT))) + 0.6 . Left ve)) + 0.6 , 25. Thu2.7) with an RWT that is ≤ 0.41 or > 0.41, respectively. We defined left ventricular hypertrophy as a LVMI ≥ 38.6 g/m2.7 and further specified this as eccentric left ventricular hypertrophy (eLVH) if RWT was ≤ 0.41 and concentric left ventricular hypertrophy when RWT was > 0.41.Cardiac geometry was further specified using the relative wall thickness (RWT) and LVMI, according to the method of Ganau et al., where RWT was calculated as 2*LVPWT/LVEDD . In chilt test for numerical variables and χ2 or Fisher’s exact test for categorical variables. Multivariable linear regression analysis was used to evaluate the association between several relevant demographic, anthropometric, and comorbidity endpoints and LVMI, correcting for BMI z score. As sensitivity analysis, these analyses were repeated, where we additionally corrected for age and gender. The associations between the dependent and independent variables were assessed separately as the sample size was too small to include all independent variables in one regression model. All assumptions were checked using plots , where Cook’s distance > 1 was used to define an influential outlier. In case normality was violated, a log or square-root transformation was considered to achieve normality. IBM SPSS Statistics for Windows  was used for the aforementioned statistical analyses. Two-sided p-value ≤ 0.05 was considered statistically significant.Numerical data are presented as mean ± standard deviation and range where appropriate. Categorical data are presented as number (percentage). Demographic and clinical variables were compared between patients with no LVH and patients with LVH, using an independent samples Of the 60 adolescents included in the BASIC trial, 43 subjects received a baseline cardiac ultrasound, of whom 42 were performed by CvL. Seventeen patients did not receive an ultrasound due to logistic circumstances, mainly unavailability of a pediatric cardiologist.2, with a mean BMI z score of 3.5 ± 0.3.The baseline characteristics of the 43 remaining adolescents are presented in Table 2.7 (± 8.3), IVST of 9.6 mm (± 1.3), LVPWT of 9.1 mm (± 1.0), and LVEDD of 50.3 mm (± 4.7). The average RWT was 0.37 (± 0.05). Twenty-four patients (55.8%) had a LVMI of ≥ 38.6 g/m2.7, corresponding to LVH according to the pediatric definition [The mean values measured with cardiac ultrasonography in our study population were: LVM of 170.4 g (± 42.3), LVMI of 40.4 g/mfinition . Figure 2.7), upon which only six patients (14.0%) would have LVH with no statistically significant differences between the group with LVH and without LVH.Differences in demographic, anthropometric, and potentially pathogenic factors between patients with LVH and without LVH are shown in Tables z score, showed that increased LVMI was associated with higher AHI  as well as sqrtAHI  after correction for BMI z score. The association of AHI and sqrtAHI with LVMI persisted after further correcting for age and sex in addition to BMI z score .By contrast, multivariable linear regression analysis, correcting for BMI B = 0.005; 95% CI 0.002 to 0.007), higher HOMA-IR , lower HDL-c , higher triglyceride levels , and higher AHI , independent of BMI z score. After further correcting for age and gender, significance persisted for AHI, HOMA-IR, and fasting insulin levels.Furthermore, we found that higher IVST was associated with a number of cosevere risk factors, namely higher fasting insulin (The major findings from our study population of severely obese adolescents are as follows: (1) LVH is highly prevalent and is mostly of the eccentric type; (2) higher LVMI is not significantly associated with anthropometric measures but is independently associated with male gender and higher AHI, the latter implying a pathophysiological relationship between nocturnal hypoxemia and LVH; (3) higher IVST is associated with male gender, lower insulin sensitivity, adverse lipid profile, and higher AHI, independent of BMI z score, which suggests that IVST is associated with an adverse metabolic profile in severely obese adolescents.. describe a similar prevalence of abnormal left ventricular geometry of 64% in their population of 13–19 year old patients with a mean BMI of 60 ± 9 kg/m2, though they used the adult cut-off of the LVMI of 51 g/m2.7 [. had on average an even higher BMI than our population (15.8 kg/m2 difference). We recommend that future studies evaluate more specifically defined populations and determine left ventricular geometry by utilization of LVMI (g/m2.7) and RWT, using the pediatric 95th percentile cut-off values of 38.6 g/m2.7 and 0.41 cm, respectively, or reporting results using both the pediatric and adult cut-off values [Multiple studies describing geometrical changes in obese children have been performed, though they are limited by a high level of intra- and interstudy variability in population characteristics  and methodology , resulting in a reported prevalence of LVH ranging from 10 to 46% , while of values , 25, 27.. described a significant association between LVM and body fat percentage [2 (± 5.5), we did not find an association between LVH and any anthropometric variable, such as BMI, neck circumference, and abdominal circumference. Our findings by them self could suggest that in this population at high risk for LVH, additional anthropometric measurements are not valuable to stratify risk. However, the findings by Ippisch et al. raise the question whether further increases in BMI are associated with an increased severity of left ventricular hypertrophy [The association between anthropometric measures, in particular BMI and abdominal circumference, with metabolic syndrome is well established . Similarrcentage . Within ertrophy .Our hypothesis that higher LVM could be independently associated with an adverse metabolic profile was based on the adult literature that shows an independent association between higher LVM, insulin resistance, and dyslipidemia –39. We dWe did not find an association between blood pressure and any parameter of left ventricular geometry , possibly because the levels of systolic and diastolic blood pressure in our population were relatively normal, with z scores of 0.7 (± 1.0) and 0.3 (± 0.8), respectively. These findings are in conjunction with the observations of the Bogalusa heart study, which found that increased left ventricular mass in obese children and young adults preceded the development of increases in systolic and diastolic blood pressure . This fu. demonstrated that treatment of OSA in adults resulted in a reduction in interventricular septal thickness [. found that in children and adolescents, an AHI > 10 was associated with a significantly higher risk of LVH when compared to primary snorers  [. evaluated children between 6 and 13 years old with or without OSA and found that higher AHI values were associated with increased IVST and RWT [. compared the prevalence of LVH in 61 subjects with or without OSA, with a mean age of 13.1 years and a median BMI of 36.3 kg/m2 [OSA is a well-established independent risk factor for LVH in adults, both through hemodynamic and nonhemodynamic mechanisms . Clowardhickness . Amin et 1.9–64) . Chan et and RWT . Further and RWT . Recentl.3 kg/m2 . Our finThe main strengths of our study are a high level of methodological standardization and extensive evaluation across several relevant clinical domains. Our study population consists of well-defined prospectively recruited severely obese adolescents aged 14–16 years, allowing adequate characterization of this specific population. We were able to evaluate multiple hypotheses related to the determinants of LVH in severely obese adolescents, owing to the extensive data we have on the overall condition of our study population, including hemodynamic factors, metabolic aberrations, and polysomnographic characteristics.Our study has a number of limitations. A significant number of patients did not receive cardiac ultrasound due to logistic reasons, resulting in a limited sample size. This may have hampered the statistical significance of certain findings and did not allow us to investigate all risk factors in one model, i.e., correcting for the influence of these factors on each other. Moreover, our population concerns mainly females, and therefore, the applicability of these findings to male severely obese adolescents cannot be guaranteed.We conclude that LVH, especially eLVH, occurs in the majority of severely obese adolescents and that adverse geometrical changes, in particular increased IVST, are independently associated with multiple commonly occurring nonhemodynamic risk factors, including OSA, dyslipidemia, and insulin resistance. We recommend low threshold of suspicion of these risk factors and proactive treatment, in order to potentially improve cardiovascular prognosis. Future research should focus on the effect of reversal of these risk factors on cardiac geometry.


Background: Numerous studies have used autologous mesenchymal stem cell injections (AMSCI) to treat osteoarthritis. We hypothesized that AMSCI is an effective osteoarthritis treatment with increasing efficacy at higher doses. Methods: We conducted a PubMed search for human clinical studies using AMSCI for the treatment of osteoarthritis (OA) and a second search for placebo arms of injectate OA treatment. Inclusion criteria included treatment outcomes ratings both pre-treatment and at least 6 months post-treatment. Results: 45 AMSCI cohorts from 34 studies met criteria. All AMSCI cohorts showed improvement at mean 15.3 months post-treatment. Mean WOMAC and VAS scores improved at 6-months and at final follow-up . Scores > 2 years were also significant (WOMAC p = 0.001/VAS p = 0.004). Results greatly exceeded the minimal clinically important difference (MCID) at each time point. AMSCI improvement also substantially exceeded previously published 6-month placebo-treatment improvement. No dose–response relationship was seen. AMSCI cohorts showed continuing improvement ≥ 6 months, and continued upward at one year. Placebo scores were already trending downward by 6 months. Conclusions: AMSCI is a consistently significantly effective treatment for osteoarthritis. It should no longer be stated that data is insufficient to establish AMSCI efficacy for OA. Given its excellent safety profile, AMSCI should be widely used for the treatment of osteoarthritis. Osteoarthritis afflicts tens of millions of people worldwide, resulting in tremendous morbidity and economic costs . The mosThe intra-articular injection of autologous mesenchymal stem cells (AMSCI) has been used in several studies for the treatment of knee osteoarthritis. AMSCIs are used in several forms including stromal vascular fraction (SVF), culture expanded adipose derived stem cells (ASCs), minimally manipulated fat graft (MM Fat), bone marrow aspirate (BMAC), and culture expanded bone marrow (BMSCs). A review of the literature has shown that they are completely safe in this application . If effeWe performed a comprehensive literature search using the PubMed database for the treatment of knee osteoarthritis with mesenchymal stem cells (MSCs) in humans, using the terms: “osteoarthritis/arthrosis”, “stem cell”, “mesenchymal stem cell”, “MSC”, “stromal vascular fraction”, “SVF”, “minimally manipulated fat”, “micro-fragmented fat”, “adipose-derived stem cell”, “bone marrow derived stem cell”, “cultured stem cell”, and “bone marrow aspirate”. In addition, we reviewed the references of the articles found this way to find other articles potentially matching our criteria. All papers found were read thoroughly, and papers were included in the study if they reported clinical outcome data pre-treatment and at least six months post-treatment. The included papers were separated into two groups, those that included pre- and post- mean Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and/or pre- and post- mean visual analogue scale (VAS) scores of patient self-reported global pain to evaluate outcomes (Group 1), and those that used other evaluation outcome ratings (Group 2). Papers were excluded that combined stem cell treatment with surgery or immunosuppression, used hematopoietic or allogeneic stem cells, or did not use joint injection as the treatment approach see .We performed a second comprehensive literature search of PubMed for randomized clinical trials in adult humans of therapy for osteoarthritis of the knee which included a single injection or up to three injections of ‘normal’ or physiological saline (0.9% NaCl) or phosphate-buffered saline (PBS) as a placebo cohort. The search was performed for the keywords “knee”, “placebo”, and “arthritis”, as well as any one of the following keywords for an intra-articular injective therapy for osteoarthritis: “Adant”, “Arthrease”, “Arthrum”, “Artz”, “Artzal”, “Biohy”, “Clodronate”, “cortisone”, “Durolane”, “Euflexxa”, “Fermathron”, “Gel-200”, “Gel-one”, “Gelsyn-3”, “Genvisc”, “Go-on”, “Hya-ject”, “Hya-joint”, “hyalgan”, “hyaluronate, hyaluronic acid”, “Hylan g-f 20”, “Hymovis”, “Interleukin-1 receptor antagonist”, “lmwf-5a”, “Monovisc”, “Nasha”, “Nrd-101”, “Nuflexxa”, “Orthovisc”, “Ostenil”, “platelet-rich plasma”, “Replasyn”, “Slm-10”, “sodium hyaluronate”, “steroid”, “steroidal”, “Structovial”, “Sunevyl”, “Supartz”, “Suplasyn”, “Synject”, “Synovial”, “Synvisc”, “Synvisc-one”, “tgf-ß1-expressing chondrocytes”, “triamcinolone acetonide”, “Variofill”, or “Zeel compositum”. Studies were discarded if they were not written in English and if the injective therapy was paired with another intervention, including physical therapy programs and surgical procedures. Studies were excluded if they did not include at least a 3-month follow-up. Similarly to the AMSCI studies, only studies that included WOMAC and/or VAS pre- and post-treatment scores were included. This group was labeled as Group 3.Group 1 papers were evaluated, and the follow-up duration and WOMAC and VAS scores pre- and post-treatment were noted. Mean scores were calculated for pre-treatment, at 6 months post-treatment, maximum change post-treatment, and final change post-treatment. For the WOMAC score, all papers were converted to a 0–96-point scale, and for the VAS, all scores were converted to a 0–100 point scale. All other reported result intervals were noted. Given the variability in study protocols, each study was weighted equally in calculating these means.Group 2 papers were evaluated for the outcome measures that were used and the outcomes that were reported.All the included papers were categorized based on the type of MSC preparation used. Each subgroup was individually analyzed.Group 3 papers were evaluated in the same manner as Group 1 papers, and the follow-up duration and WOMAC and VAS scores pre- and post-treatment were noted. Mean scores were calculated for pre-treatment, at 3 months post-treatment, and at 6 months post-treatment, where available. Peak time of effect was noted. WOMAC scores were converted to a 0–96-point scale and VAS scores were converted to a 0–100 point scale as needed. All other reported result intervals were noted. Given the variability in study protocols each study was weighted equally in calculating these means.Our search yielded 34 studies published in peer-reviewed journals that examined the injection of autologous AMSCI for the treatment of knee osteoarthritis and met our criteria for rating evaluation. Of these papers, 29 studies included mean WOMAC and/or VAS scores (Group 1) and 5 did not (Group 2) see . Our secThe 29 studies in Group 1 included 1063 treated knees. Six of the studies included two or more arms with different AMSCI treatments, resulting in 38 treatment arm subgroups included in this analysis. Twelve of the subgroups were treated with ASCs, 7 were treated with SVF, 7 were treated with BMAC, 6 were treated with BMSCs, and 4 were treated with MM Fat, and 2 were treated with BMAC & MM Fat. The number of cells used in each treatment is listed in p < 0.0001 for all).Twenty-six of the study subgroups included WOMAC scores, with 6-month follow-up WOMAC scores being available for 22 study subgroups. The mean pre-treatment WOMAC score of these studies was 45.1 . The mean WOMAC score decreased at the six-month follow-up to 25.9 and to 20.3 at the final follow-up point, for a total change in score of 23.0 and 25.2, respectively. Comparison of means was performed between pre-treatment scores and post-treatment scores at 6 months and final follow-up, and were significantly different at all follow-up points .Thirty-two study subgroups included VAS scores. Six-month VAS scores were available for 27 study subgroups. The mean pre-treatment VAS score was 56.2 . The mean VAS score decreased to 29.6 at six months and 27.1 at the final follow-up point, for a total change in score of 28.7 and 28.7. Comparison of means was performed between pre-treatment scores and post-treatment scores at 6 months and final follow-up and was significantly different from pre-treatment to all follow points .Six study arms of four studies in Group 1 reported changes in WOMAC scores for 6 months, 1 year and some point longer than 1 year ,30,37,43The five studies in Group 2 had seven subgroups which included 193 joints and used a variety of other outcome scores See  Lamo-Espp = 0.04, 6 mo p = 0.07). VAS scores were not statistically different from pre-treatment to post-treatment at either time point . Comparison of means was performed comparing the change in mean at 6 months between Group 1 and Group 3. The means of the two groups were statistically different for both WOMAC (p < 0.0001) and VAS (p = 0.003).There were 18 studies, one with two placebo arms, in Group 3, resulting in 19 groups with a total of 1793 patients see . Sixteenp = 0.02 at ≤3 months, p < 0.0001 at 6 months). The VAS scores are similar, except that the Group 3 scores are never above the MCID. Comparison of means between the Group 1 and placebo VAS scores is significantly different at all points .The mean WOMAC and mean VAS scores for Group 1 and Group 3 studies that reported a 6-month score and at least 1 endpoint prior to 6 months are shown in A comparison was made using Groups 1 and 2 based on the type of AMSCI used. BMAC, BMAC/MM Fat and MM Fat groups were excluded because of low reporting of WOMAC scores in these groups with only three reported results in the MM Fat group, two in the BMAC group and none in the BMAC+ MM Fat group. For the remaining three groups , a MCID ratio was calculated for each study at 6 months and at final follow-up. For Group 1 studies and the Group 2 study that used WOMAC scores, the MCID ratio was calculated as the reported WOMAC score divided by the WOMAC MCID of 11.0. For the Group 2 studies that reported KOOS scores, the MCID ratio was the sum of the scores from the five subgroups divided by 5 and then divided by the KOOS MCID of 10. The MCID ratios can be found in A comparison was performed to see if a dose–response relationship existed between numbers of cells and outcome parameters. For each of the three groups, ASCs BMSCs, and SVF, the MCID ratio at final follow-up was compared to the total number of cells used in treatment. For ASC and BMSC groups, the total number of cells was based on the reported MSC cells. For the SVF group, the reported total nucleated cells (TNCs) were used. A regression analysis was performed. The results are graphed in This is the first paper to examine the spectrum of autologous mesenchymal stem cell treatment in the treatment of osteoarthritis for efficacy. It is the first to show consistent clinically important efficacy from these treatments. It is also the first to examine dose–response curves for these treatments and demonstrate the lack of same so far.All 26 of the Group 1 subgroups which included WOMAC scores showed improvement from pre-treatment scores to final follow-up. These improvements were statistically significant at all time points. Treatment with AMSCI produced, on average, strong improvement in WOMAC scores that continued to improve over time, with one year follow-up showing greater improvement than 6-month follow-up.In contrast, the Group 3 placebo WOMAC mean change peaked before 3 months and was 9.0 at 6 months and falling. Although the scores were both statistically improved and slightly above the MCID initially, showing some placebo or saline effect, the results fell off quickly and by 6 months were well below the MCID and clinically insignificant. These results demonstrate that the placebo effect peaks early and is not large. The effects of treatment seen in the Group 1 results are larger, more sustained, and continue to rise over time, showing a definite positive effect of treatment well beyond the placebo effects of saline injection.Similarly, all 23 Group 1 study subgroups that included VAS scores showed improvement from pre-treatment to all short, intermediate, and long term follow-up points. These improvements were statistically significant at all time points.The Group 3 placebo studies had a mean change in VAS scores substantially below the MCID at all time points. Only three of the studies exceeded the MCID at any point in time. VAS scores peaked early (about 2 months) and the results declined from there. The follow-up results were not statistically different from the pre-treatment scores at any point. Similarly to the WOMAC scores, the change in VAS scores demonstrate a small placebo effect and a much larger improvement in Group 1, which indicates significant improvement from treatment with AMSCI.In the case of both the WOMAC and VAS scores and as seen in Of the six different types of AMSCI treatments in this study, three of them  had too few cohorts with WOMAC or KOOS scores to allow meaningful comparison. In the other three groups , the change in scores was very similar between the groups at both 6 months and final follow-up.It was hypothesized that there would be a positive relationship between cell number and improved outcome. However, no dose–response relationship between cell dose and outcome was found, refuting this part of our hypothesis. This was true for both MSCs and SVF, which measures TNC. This universally beneficial effect of AMSCI across widely varying doses indicates that there may be a threshold effect demonstrated, but not a dose–response relationship. A meta-analysis of mesenchymal stem cells from Cui  also fouThe presences or absence of a dose–response curve is a matter of some importance, because increased dosing of cultured cells engenders greater cost. Increased dosing of stromal vascular fraction or bone marrow aspirate increases the time and discomfort of the procedure. If smaller doses have equal efficacy to larger ones, the cost and difficulty of AMSCI procedures could be better controlled. The addition of data from future studies should help to settle this question and generate optimal dosing regimens and perhaps identify a minimally effective cell dose.Limitations of this study are the limited number of cohorts available for analysis. However, the fact that every one of the 45 treatment subgroups showed clinical efficacy is more than a sufficient number to validate our hypothesis of consistent AMSCI efficacy. The heterogeneity of the studies also limited our ability to compare treatment types.These results show that AMSCI treatment has, arguably, the greatest proven efficacy of any known treatment for osteoarthritis. The treatment is longer lasting than other non-surgical treatments and has the best safety profile. Joint replacement carries significant morbidity and mortality, and often produces poorer outcomes than expected by patients . HA and Up until now, discussions of stem cell treatments have generally begun with a disclaimer that they are certainly “promising”, but that there is “insufficient data” to determine if they are effective or safe. We believe that this study conclusively refutes this agnosticism and changes that narrative. In fact, the results reported here show that there is more than enough data to conclude that autologous mesenchymal stem cell treatment, while not a “cure”, is clearly consistently significantly effective for OA. Our companion study  shows thThis study demonstrates that autologous mesenchymal stem cell injection provides consistent clinically important improvement in OA disease parameters, and that both the magnitude and the duration of improvement far exceed any improvement that may be seen from a placebo effect and is well above the MCID. Furthermore, the duration of efficacy out to two years from a single treatment greatly exceeds that of any other nonsurgical treatment or any possible placebo effect. Particularly in light of its excellent safety profile and the absence of other safe effective treatments for osteoarthritis, AMSCI treatment should be widely adopted for the treatment of osteoarthritis.