Early Transcriptional Changes of Adipose-Derived Stem Cells (ADSCs) in Cell Culture.

Background and Objectives: While autologous fat grafting has been carried out in the clinical field for many years, the utilization of isolated and cultured adipose-derived stem cells (ADSCs) is highly restricted in many countries. However, ADSCs are under investigation currently and heavily researched in many cell-based therapy approaches in the field of regenerative medicine. Objective: For the utilization of future cell-based therapies with ADSCs, in vitro cell expansion might be necessary in many cases. Thus, the cellular characteristics of ADSCs may be altered though the process of being cultured. The aim of this study was to assess changes in the gene expression profile of ADSCs after cell expansion for 48 h. Materials and Methods: Isolated ADSCs from five different donors were used for in vitro expansion. For the evaluation of the gene expression profile, mRNA deep Next-Generation Sequencing was performed to evaluate the differences between cultured and freshly isolated cells. Results: Our study gives insight into transcriptional changes in ADSCs after a short cell cultivation period. This includes the most prominent upregulated genes such as PPL, PRR15, CCL11 and ABCA9, as well the most downregulated genes, which are FOSB, FOS, EGR1 and DUSP6. Furthermore, we showed different biological processes that changed during short-term cell expansion, which led to downregulation of fat-associated metabolism hormone processes and to an upregulation of extracellular matrix-associated genes. Conclusion: In conclusion, our study reveals a detailed insight into early changes in the gene expression profile of cultured ADSCs. Our results can be utilized in future experiments.

Reduced Awareness for Osteoporosis in Hip Fracture Patients Compared to Elderly Patients Undergoing Elective Hip Replacement.

Background: Osteoporotic fractures are associated with a loss of quality of life, but only few patients receive an appropriate therapy. Therefore, the present study aims to investigate the awareness of musculoskeletal patients to participate in osteoporosis assessment and to evaluate whether there are significant differences between acute care patients treated for major fractures of the hip compared to elective patients treated for hip joint replacement.; Methods: From May 2015 to December 2016 patients who were undergoing surgical treatment for proximal femur fracture or total hip replacement due to osteoarthritis and were at risk for an underlying osteoporosis (female > 60 and male > 70 years) were included in the study and asked to complete a questionnaire assessing the awareness for an underlying osteoporosis. ASA Score, FRAX Score, and demographic information have also been examined. Results: In total 268 patients (female = 194 (72.0%)/male = 74 (28%)), mean age 77.7 years (±7.7) undergoing hip surgery were included. Of these, 118 were treated for fracture-related etiology and 150 underwent total hip arthroplasty in an elective care setting. Patients were interviewed about their need for osteoporosis examination during hospitalization. Overall, 76 of 150 patients receiving elective care (50.7%) considered that an examination was necessary, whereas in proximal femur fracture patients the awareness was lower, and the disease osteoporosis was assessed as threatening by significantly fewer newly fractured patients. By comparison, patients undergoing trauma surgery had a considerably greater risk of developing another osteoporotic fracture than patients undergoing elective surgery determined by the FRAX® Score (p ≤ 0.001).; Conclusions: The patients' motivation to endure additional osteoporosis diagnostic testing is notoriously low and needs to be increased. Patients who underwent acute care surgery for a fragility proximal femur fracture, although acutely affected by the potential consequences of underlying osteoporosis, showed lower awareness than the elective comparison population that was also on average 6.1 years younger. Although elective patients were younger and at a lower risk, they seemed to be much more willing to undergo further osteoporosis assessment. In order to better identify and care for patients at risk, interventions such as effective screening, early initiation of osteoporosis therapy in the inpatient setting and a fracture liaison service are important measures.

Self-Assessment of Mobility of People over 65 Years of Age.

BACKGROUND AND OBJECTIVES: Nowadays, various clinical scoring systems are used in the medical care of the elderly to assess the quality of mobility. However, people often tend to under- or overestimate themselves in many aspects. Since this can have serious consequences in their treatment and care, the aim of this study was to identify differences in the self and external assessment of mobility of persons over 65 years of age. MATERIALS AND METHODS: 222 participants over 65 years of age and one external, closely-related relative or professional caregiver were interviewed by a unique study assistant using a standardized questionnaire. Participants were divided into people living in nursing homes and independent people living at home, where either the caregivers or the relatives provided the external assessment of mobility, respectively. The questionnaire included demographics, cognitive abilities (Mini Mental Status Test); fall risk (Hendrich 2 Fall Risk Model); as well as the Parker Mobility Score, Barthel Index, and EQ-5D-5L to measure mobility, activities of daily life and quality of life. In each case, the participant and the external person were asked for their assessment to the participants' mobility situation. Statistical significance of the difference between self and external assessment was calculated with a Wilcoxon rank-sum test and assumed with a p-value of ≤ 0.05. RESULTS: Self-assessment indicated a significantly higher value, when compared to an external assessment for the Parker Mobility Score for females in nursing homes (p ≤ 0.01), as well as for the Barthel Index for females (p ≤ 0.01) and males (p ≤ 0.01) in nursing homes. The EQ-5D-5L received a significantly higher self-assessment value for females (p ≤ 0.01) and males (p ≤ 0.01) living at home and females (p ≤ 0.01) and males (p ≤ 0.05) in nursing homes. CONCLUSIONS: Persons over 65 years of age tend to overestimate their level of mobility, quality of life and activities of daily life. Especially for people living in nursing homes, these scoring systems should be treated with caution due to the differences between the verbal statements. It is important to properly assess the mobility situation of elderly patients to ensure correct medical treatment and prevention of falls.

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel.

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.

Tenogenic Contribution to Skeletal Muscle Regeneration: The Secretome of Scleraxis Overexpressing Mesenchymal Stem Cells Enhances Myogenic Differentiation In Vitro.

Integrity of the musculoskeletal system is essential for the transfer of muscular contraction force to the associated bones. Tendons and skeletal muscles intertwine, but on a cellular level, the myotendinous junctions (MTJs) display a sharp transition zone with a highly specific molecular adaption. The function of MTJs could go beyond a mere structural role and might include homeostasis of this musculoskeletal tissue compound, thus also being involved in skeletal muscle regeneration. Repair processes recapitulate several developmental mechanisms, and as myotendinous interaction does occur already during development, MTJs could likewise contribute to muscle regeneration. Recent studies identified tendon-related, scleraxis-expressing cells that reside in close proximity to the MTJs and the muscle belly. As the muscle-specific function of these scleraxis positive cells is unknown, we compared the influence of two immortalized mesenchymal stem cell (MSC) lines-differing only by the overexpression of scleraxis-on myoblasts morphology, metabolism, migration, fusion, and alignment. Our results revealed a significant increase in myoblast fusion and metabolic activity when exposed to the secretome derived from scleraxis-overexpressing MSCs. However, we found no significant changes in myoblast migration and myofiber alignment. Further analysis of differentially expressed genes between native MSCs and scleraxis-overexpressing MSCs by RNA sequencing unraveled potential candidate genes, i.e., extracellular matrix (ECM) proteins, transmembrane receptors, or proteases that might enhance myoblast fusion. Our results suggest that musculotendinous interaction is essential for the development and healing of skeletal muscles.

Differences in the Inflammatory Response of White Adipose Tissue and Adipose-Derived Stem Cells.

The application of liposuctioned white adipose tissue (L-WAT) and adipose-derived stem cells (ADSCs) as a novel immunomodulatory treatment option is the currently subject of various clinical trials. Because it is crucial to understand the underlying therapeutic mechanisms, the latest studies focused on the immunomodulatory functions of L-WAT or ADSCs. However, studies that examine the specific transcriptional adaptation of these treatment options to an extrinsic inflammatory stimulus in an unbiased manner are scarce. The aim of this study was to compare the gene expression profile of L-WAT and ADSCs, when subjected to tumor necrosis factor alpha (TNFα), and to identify key factors that might be therapeutically relevant when using L-WAT or ADSCs as an immuno-modulator. Fat tissue was harvested by liposuction from five human donors. ADSCs were isolated from the same donors and shortly subjected to expansion culture. L-WAT and ADSCs were treated with human recombinant TNFα, to trigger a strong inflammatory response. Subsequently, an mRNA deep nextgeneration sequencing was performed to evaluate the different inflammatory responses of L-WAT and ADSCs. We found significant gene expression changes in both experimental groups after TNFα incubation. However, ADSCs showed a more homogenous gene expression profile by predominantly expressing genes involved in immunomodulatory processes such as CCL19, CCL5, TNFSF15 and IL1b when compared to L-WAT, which reacted rather heterogeneously. As RNA sequencing between L-WAT and ADSCS treated with TNFα revealed that L-WAT responded very heterogeneously to TNFα treatment, we therefore conclude that ADSCs are more reliable and predictable when used therapeutically. Our study furthermore yields insight into potential biological processes regarding immune system response, inflammatory response, and cell activation. Our results can help to better understand the different immunomodulatory effects of L-WAT and ADSCs.

Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis.

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4-/- mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4-/- mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4-/- mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4-/- mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

Aggrecan Hypomorphism Compromises Articular Cartilage Biomechanical Properties and Is Associated with Increased Incidence of Spontaneous Osteoarthritis.

The gene encoding the proteoglycan aggrecan (Agc1) is abundantly expressed in cartilage during development and adulthood, and the loss or diminished deposition of the protein results in a wide range of skeletal malformations. Furthermore, aggrecan degradation is a hallmark of cartilage degeneration occurring in osteoarthritis. In the present study, we investigated the consequences of a partial loss of aggrecan in the postnatal skeleton and in the articular cartilage of adult mice. We took advantage of the previously described Agc1tm(IRES-CreERT2) mouse line, which allows for conditional and timely-regulated deletion of floxed, cartilage-expressed genes. As previously reported, the introduction of the CreERT2 cassette in the 3'UTR causes a disruption of the normal expression of Agc1 resulting in a hypomorphic deposition of the protein. In homozygous mice, we observed a dwarf phenotype, which persisted throughout adulthood supporting the evidence that reduced aggrecan amount impairs skeletal growth. Homozygous mice exhibited reduced proteoglycan staining of the articular cartilage at 6 and 12 months of age, increased stiffening of the extracellular matrix at six months, and developed severe cartilage erosion by 12 months. The osteoarthritis in the hypomorph mice was not accompanied by increased expression of catabolic enzymes and matrix degradation neoepitopes. These findings suggest that the degeneration found in homozygous mice is likely due to the compromised mechanical properties of the cartilage tissue upon aggrecan reduction.

The role of Sema3-Npn-1 signaling during diaphragm innervation and muscle development.

Correct innervation of the main respiratory muscle in mammals, namely the thoracic diaphragm, is a crucial pre-requisite for the functionality of this muscle and the viability of the entire organism. Systemic impairment of Sema3A-Npn-1 (Npn-1 is also known as NRP1) signaling causes excessive branching of phrenic nerves in the diaphragm and into the central tendon region, where the majority of misguided axons innervate ectopic musculature. To elucidate whether these ectopic muscles are a result of misguidance of myoblast precursors due to the loss of Sema3A-Npn-1 signaling, we conditionally ablated Npn-1 in somatic motor neurons, which led to a similar phenotype of phrenic nerve defasciculation and, intriguingly, also formation of innervated ectopic muscles. We therefore hypothesize that ectopic myocyte fusion is caused by additional factors released by misprojecting growth cones. Slit2 and its Robo receptors are expressed by phrenic motor axons and migrating myoblasts, respectively, during innervation of the diaphragm. In vitro analyses revealed a chemoattractant effect of Slit2 on primary diaphragm myoblasts. Thus, we postulate that factors released by motor neuron growth cones have an influence on the migration properties of myoblasts during establishment of the diaphragm.

Effects of medical interventions on health-related quality of life in chronic disease - systematic review and meta-analysis of the 19 most common diagnoses.

Introduction: The demographic shift leads to a tremendous increase in age-related diseases, which are often chronic. Therefore, a focus of chronic disease management should be set on the maintenance or even improvement of the patients' quality of life (QoL). One indicator to objectively measure QoL is the EQ-5D questionnaire, which was validated in a disease- and world region-specific manner. The aim of this study was to conduct a systematic literature review and meta-analysis on the QoL across the most frequent chronic diseases that utilized the EQ-5D and performed a disease-specific meta-analysis for treatment-dependent QoL improvement. Materials and methods: The most common chronic disease in Germany were identified by their ICD-10 codes, followed by a systematic literature review of these ICD-10 codes and the EQ-5D index values. Finally, out of 10,016 independently -screened studies by two persons, 538 studies were included in the systematic review and 216 studies in the meta-analysis, respectively. Results: We found significant medium to large effect sizes of treatment effects, i.e., effect size >0.5, in musculoskeletal conditions with the exception of fractures, for chronic depression and for stroke. The effect size did not differ significantly from zero for breast and lung cancer and were significantly negative for fractures. Conclusion: Our analysis showed a large variation between baseline and post-treatment scores on the EQ-5D health index, depending on the health condition. We found large gains in health-related quality of life mainly for interventions for musculoskeletal disease. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020150936, PROSPERO identifier CRD42020150936.

Detailed analysis of surgically treated hand trauma patients in a regional German trauma centre.

Hand and forearm injuries are the most frequent reason for consultations in German emergency departments. Therefore, full recovery has a high social and economic relevance. In this study, data on surgically treated hand injuries in a regional German trauma centre between 01.01.2019 and 31.01.2021 were collected using the new German HandTraumaRegister of the German Society for Hand Surgery. These data were retrospectively analysed and correlated with mobility data of the Bavarian population, the 7-day incidence of Covid-19 infections in Germany and the number of elective hand surgeries. We found that a fall from standing height with consecutive distal radius fracture was the most common injury in women, whereas mechanism of injury and diagnosis were more diverse in men. The populations' mobility correlated well with the number of accidents, which in turn was reciprocal to the 7-day-incidence of Covid-19 infections. The number of elective hand surgeries expectedly dropped significantly during the state-imposed lockdowns. Knowing that mainly young men and elderly women suffer from hand injuries, tailored prevention measures may be elaborated. In order to reduce socioeconomic burden, care for hand injuries and elective hand surgeries must be guaranteed according to the frequency of their occurrence.

Development of a Machine Learning-Based Model to Predict Timed-Up-and-Go Test in Older Adults.

INTRODUCTION: The measurement of physical frailty in elderly patients with orthopedic impairments remains a challenge due to its subjectivity, unreliability, time-consuming nature, and limited applicability to uninjured individuals. Our study aims to address this gap by developing objective, multifactorial machine models that do not rely on mobility data and subsequently validating their predictive capacity concerning the Timed-up-and-Go test (TUG test) in orthogeriatric patients. METHODS: We utilized 67 multifactorial non-mobility parameters in a pre-processing phase, employing six feature selection algorithms. Subsequently, these parameters were used to train four distinct machine learning algorithms, including a generalized linear model, a support vector machine, a random forest algorithm, and an extreme gradient boost algorithm. The primary goal was to predict the time required for the TUG test without relying on mobility data. RESULTS: The random forest algorithm yielded the most accurate estimations of the TUG test time. The best-performing algorithm demonstrated a mean absolute error of 2.7 s, while the worst-performing algorithm exhibited an error of 7.8 s. The methodology used for variable selection appeared to exert minimal influence on the overall performance. It is essential to highlight that all the employed algorithms tended to overestimate the time for quick patients and underestimate it for slower patients. CONCLUSION: Our findings demonstrate the feasibility of predicting the TUG test time using a machine learning model that does not depend on mobility data. This establishes a basis for identifying patients at risk automatically and objectively assessing the physical capacity of currently immobilized patients. Such advancements could significantly contribute to enhancing patient care and treatment planning in orthogeriatric settings.

One bout of endurance exercise does not change gene expression or proliferation in a C26 colon carcinoma in immunocompetent mice.

PURPOSE: Exercise typically reduces tumour growth, proliferation and improves outcomes. Many of these effects require exercise to change gene expression within a tumour, but whether exercise  actually affects gene expression within a tumour has not been investigated yet. The aim of this study was, therefore, to find out whether one bout of endurance exercise alters gene expression and proliferation in a C26 carcinoma in immunocompetent mice. METHODS: BALB/c were injected with C26 colon carcinoma cells. Once the tumours had formed, the mice either ran for 65 min with increasing intensity or rested before the tumour was dissected. The tumours were then analysed by RNA-Seq and stained for the proliferation marker KI67. RESULTS: One bout of running for 65 min did not systematically change gene expression in C26 carcinomas of BALB/c mice when compared to BALB/c mice that were rested. However, when analysed for sex, the expression of 17, mostly skeletal muscle-related genes was higher in the samples of the female mice taken post-exercise. Further histological analysis showed that this signal likely comes from the presence of muscle fibres from the panniculus carnosus muscle inside the tumours. Also, we found no differences in the positivity for the proliferation marker KI67 in the control and exercise C26 carcinomas. CONCLUSION: A bout of exercise did not systematically affect gene expression or proliferation in C26 carcinomas in immunocompetent BALB/c mice.

Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression.

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), ß(1) and ß(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.

Prediction of Physical Frailty in Orthogeriatric Patients Using Sensor Insole-Based Gait Analysis and Machine Learning Algorithms: Cross-sectional Study.

BACKGROUND: Assessment of the physical frailty of older patients is of great importance in many medical disciplines to be able to implement individualized therapies. For physical tests, time is usually used as the only objective measure. To record other objective factors, modern wearables offer great potential for generating valid data and integrating the data into medical decision-making. OBJECTIVE: The aim of this study was to compare the predictive value of insole data, which were collected during the Timed-Up-and-Go (TUG) test, to the benchmark standard questionnaire for sarcopenia (SARC-F: strength, assistance with walking, rising from a chair, climbing stairs, and falls) and physical assessment (TUG test) for evaluating physical frailty, defined by the Short Physical Performance Battery (SPPB), using machine learning algorithms. METHODS: This cross-sectional study included patients aged >60 years with independent ambulation and no mental or neurological impairment. A comprehensive set of parameters associated with physical frailty were assessed, including body composition, questionnaires (European Quality of Life 5-dimension [EQ 5D 5L], SARC-F), and physical performance tests (SPPB, TUG), along with digital sensor insole gait parameters collected during the TUG test. Physical frailty was defined as an SPPB score≤8. Advanced statistics, including random forest (RF) feature selection and machine learning algorithms (K-nearest neighbor [KNN] and RF) were used to compare the diagnostic value of these parameters to identify patients with physical frailty. RESULTS: Classified by the SPPB, 23 of the 57 eligible patients were defined as having physical frailty. Several gait parameters were significantly different between the two groups (with and without physical frailty). The area under the receiver operating characteristic curve (AUROC) of the TUG test was superior to that of the SARC-F (0.862 vs 0.639). The recursive feature elimination algorithm identified 9 parameters, 8 of which were digital insole gait parameters. Both the KNN and RF algorithms trained with these parameters resulted in excellent results (AUROC of 0.801 and 0.919, respectively). CONCLUSIONS: A gait analysis based on machine learning algorithms using sensor soles is superior to the SARC-F and the TUG test to identify physical frailty in orthogeriatric patients.

SFRP2 Overexpression Induces an Osteoblast-like Phenotype in Prostate Cancer Cells.

Prostate cancer bone metastasis is still one of the most fatal cancer diagnoses for men. Survival of the circulating prostate tumor cells and their adaptation strategy to survive in the bone niche is the key point to determining metastasis in early cancer stages. The promoter of SFRP2 gene, encoding a WNT signaling modulator, is hypermethylated in many cancer types including prostate cancer. Moreover, SFRP2 can positively regulate osteogenic differentiation in vitro and in vivo. Here, we showed SFRP2 overexpression in the prostate cancer cell line PC3 induces an epithelial mesenchymal transition (EMT), increases the attachment, and modifies the transcriptome towards an osteoblast-like phenotype (osteomimicry) in a collagen 1-dependent manner. Our data reflect a novel molecular mechanism concerning how metastasizing prostate cancer cells might increase their chance to survive within bone tissue.

Bupivacaine, ropivacaine, and morphine: comparison of toxicity on human hamstring-derived stem/progenitor cells.

PURPOSE: Bupivacaine, ropivacaine, and morphine are commonly administered intraarticularly after anterior cruciate ligament (ACL) reconstruction. However, their effects on human tendon stem/progenitor cells (TSPC) have not been studied. Therefore, this study investigates the cytotoxicity of these analgetics on TSPC. METHODS: Cells were isolated from human hamstring grafts of 3 female (age 15, 16 and 59) and 2 male patients (age 16 and 47). Cells were incubated using 0.5% bupivacaine, 0.5/0.75% ropivacaine, and 0.025% morphine. Cell viability was assessed after 0.5, 2, and 6 h using live/dead assay. Metabolic activity and apoptosis were measured by WST- and Annexin-V-FACS-assay after 2 h. RESULTS: Cell viability remained unchanged after 0.5 h in all groups, while treatment with bupivacaine and 0.5/0.75% ropivacaine resulted in a complete cell loss after 6 h. Contrarily, morphine showed no cytotoxic effect. Cell viability and metabolism were significantly reduced after treatment with bupivacaine (22.1; 8.3%) and 0.75% ropivacaine (56.5; 23.8%), while 0.5% ropivacaine and morphine showed no significant difference compared with controls. Apoptosis was significantly induced after incubation with bupivacaine (58.1%) and 0.75% ropivacaine (26.2%), whereas 0.5% ropivacaine only led to a slight induction compared with morphine and controls. CONCLUSIONS: Clinically administered concentrations of bupivacaine (0.5%) and ropivacaine (0.75%) have a significant cytotoxic effect on human TSPC in vitro, while ropivacaine in a concentration of 0.5% has a mild but not significant effect on apoptosis and cell metabolism. In contrast, morphine does not affect cell survival, metabolism, or apoptosis. Knowing that morphine provides comparable to even prolonged pain reduction after ACL reconstruction, the presented in vitro study suggests morphine as a potentially less toxic analgetic drug for intraarticular application in clinical practice.

Hypoxic Signaling in Skeletal Muscle Maintenance and Regeneration: A Systematic Review.

In skeletal muscle tissue, oxygen (O2) plays a pivotal role in both metabolism and the regulation of several intercellular pathways, which can modify proliferation, differentiation and survival of cells within the myogenic lineage. The concentration of oxygen in muscle tissue is reduced during embryogenesis and pathological conditions. Myogenic progenitor cells, namely satellite cells, are necessary for muscular regeneration in adults and are localized in a hypoxic microenvironment under the basal lamina, suggesting that the O2 level could affect their function. This review presents the effects of reduced oxygen levels (hypoxia) on satellite cell survival, myoblast regeneration and differentiation in vertebrates. Further investigations and understanding of the pathways involved in adult muscle regeneration during hypoxic conditions are maybe clinically relevant to seek for novel drug treatments for patients with severe muscle damage. We especially outlined the effect of hypoxia-inducible factor 1-alpha (HIF1A), the most studied transcriptional regulator of cellular and developmental response to hypoxia, whose investigation has recently been awarded with the Nobel price.

Adipose-derived stem/progenitor cells from lipoaspirates: A comparison between the Lipivage200-5 liposuction system and the Body-Jet liposuction system.

BACKGROUND: Adipose-derived stem/progenitor cells (ADSPCs) are under investigation in many clinical applications for their regenerative potential in a variety of autoimmune, degenerative, and inflammatory diseases. Adipose tissue, which is mainly harvested by manual liposuction, is the main source of these ADSPCs. OBJECTIVE: In the past years, a variety of different liposuction devices have been commercialized. To ensure a high quality of obtained ADSPCs, it is crucial to show the advantages and disadvantages of frequently used liposuction methods. For this reason, the objective of this study was to compare ADSPCs harvested by either the suction-assisted LipiVage200-5 or the water-assisted Body-Jet system. METHODS: The proliferation potential of ADSPCs, harvested from 20 patients, was assessed by cumulative population doublings (cumPD), population doubling time (PDT), colony-forming units (CFU), and cell metabolism assays. To prove the multipotency of the primary isolated cells, ADSPCs were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages. RESULTS: Our data show a significantly higher cumPD, as well as a significantly lower PDT for cells obtained by the Body-Jet system. No significant differences were found regarding the CFU efficiency and the cell metabolism. Furthermore, we showed that the adipogenic, osteogenic, and chondrogenic potential of ADSPCs is similar in both groups. DISCUSSION/CONCLUSION: In our study, we provide evidence that the cell characteristics and the functional properties of ADSPCs isolated after liposuction with different techniques are largely similar. However, we observed a significantly higher cumPD and a slightly higher adipogenic potential in cells isolated after liposuction with the Body-Jet system. Different cannula sizes and sheer stresses in the used methods might play a role here.

Fibrin glue displays promising in vitro characteristics as a potential carrier of adipose progenitor cells for tissue regeneration.

Adipose-derived multipotent stem/progenitor cells (ASPCs) were shown to be ideal candidates for cell-based regenerative therapies. Yet, despite their huge potential, successful clinical applications are still rare. It was suggested that the efficacy of ASPCs at the recipient site depends on the vehicle of cell delivery. In this study, for preparation of a murine critical-size nerve defect model, we assessed the commercially available fibrin gel (ARTISS) as a potential cell carrier. In a thorough in vitro analysis, we investigated cell-fibrin interactions and analyzed the distribution and the long-term behavior of ASPCs cultivated in fibrin gel under normoxic and hypoxic conditions. ASPCs attached to the surface of a thin fibrin layer (two-dimensional condition) and spread with the abundant formation of actin stress fibers. Cells cultured within a fibrin matrix (three-dimensional condition) displayed a uniform distribution and formed interconnected networks while exhibiting strong cell-matrix interactions. Using time-lapse analysis, cells were found to migrate out of the gel and subsequently proliferated robustly both under hypoxic and normoxic conditions. During 14 days of culture in fibrin gel, ASPCs showed high viability, metabolic, and remodeling activities. At the end of the culture period, the fibrin matrix was degraded entirely accompanied by an upregulation of matrix metalloproteinases. In conclusion, fibrin gel stands out as a valuable biomaterial for delivering vital and active cells to damaged tissues. As a direct proof, ASPCs carried in a fibrin matrix will be evaluated in a murine critically sized peripheral nerve repair model.