RNA-seq unravels distinct expression profiles of keloids and Dupuytren's disease.

Keloid scars and Dupuytren's disease are two common, chronic, and incurable fibroproliferative disorders that, among other shared clinical features, may induce joint contractures. We employed bulk RNA sequencing to discern potential shared gene expression patterns and underlying pathological pathways between these two conditions. Our aim was to uncover potential molecular targets that could pave the way for novel therapeutic strategies. Differentially expressed genes (DEGs) were functionally annotated using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The protein-protein-interaction (PPI) networks were constructed by using the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape. The Molecular Complex Detection (MCODE) plugin was used for downstream analysis of the PPI networks. A total of 1922 DEGs were identified within Dupuytren's and keloid samples, yet no overlapping gene expression profiles were detected. Significantly enriched GO terms were related to skin development and tendon formation in keloid scars and Dupuytren's disease, respectively. The PPI network analysis revealed 10 genes and the module analysis provided six protein networks, which might play an integral part in disease development. These genes, including CDH1, ERBB2, CASP3 and RPS27A, may serve as new targets for future research to develop biomarkers and/or therapeutic agents.

Systematic review of molecular pathways in burn wound healing.

Depending on extent and depth, burn injuries and resulting scars may be challenging and expensive to treat and above all heavily impact the patients' lives. This systematic review represents the current state of knowledge on molecular pathways activated during burn wound healing. All currently known molecular information about gene expression and molecular interactions in mammals has been summarized. An ample interaction of regenerative cytokines, growth factors, ECM-regenerative molecules and proinflammatory immune response became apparent. We identified three molecules to be most often involved in the pathways: TGFB1, ACTA1 and COL1A1. Yet, other factors including FLII, AKT1 and miR-145 were shown to play pivotal roles in burn wound healing as well. This systematic review helps to explain the fundamental molecular proceedings participating in burn wound healing. A number of new molecular interactions and functional connections were identified yielding intriguing new research targets. An interactive version of the first network about molecular pathways and interactions during burn wound healing is provided in the online edition and on WikiPathways.

The influence of vitamin D on handgrip strength in elderly trauma patients.

BACKGROUND AND OBJECTIVES: The treatment of elderly patients is an increasing challenge and the long-term sequelae often affect activities of daily living and quality of life in those patients. Handgrip strength (HGS) appears as a promising value to predict the outcome after trauma in elderly patients and to assess the overall muscle strength. Besides the possible role of psychological and hormonal factors, vitamin D may have a positive influence. Furthermore, some data suggest that Vitamin D is beneficial regarding muscle strength and possibly prevents further falls and injuries in orthogeriatric patients. The purpose of this study was to identify if Vitamin D is an influencing factor for HGSin elderly trauma patients. MATERIALS AND METHODS: 94 elderly patients in a Level I Trauma Center aged 60 years or older were prospectively enrolled and HGS as well as serum 25-OH Vitamin D concentration (VDC) were measured. In addition, the standardized questionnaires Barthel Index (BI), Parker Mobility Score (PMS), Short Physical Performance Battery (SPPB), Strength, Assistance with walking, Rise from a chair, Climb stairs and Falls (SARC-F) and European Quality of Life 5 Dimensions 5 Levels Questionnaire (EQ-5D-5L), were used to record mental health status and demographic data. RESULTS: HGS is mainly related to age and sex in elderly trauma patients. HGS was higher in men (meanmale = 27.31 kg (± 8.11), meanfemale = 15.62 kg (± 5.63), p < 0.001 and decreased with age (ßage = - 0.58, p < 0.001). A significant negative correlation between HGS and VDC exists in the overall sample (ßVDC = - 0.27, pVDC < 0.008), which still remains after adjusting for age (pVDC < 0.004), but is not significant after adjustment for both main confounders, age and sex (pVDC < 0.08). Furthermore, the HGS was lower in pateints who reported frequent falls, stumbling, dizziness or a late onset of menopause, and decreased if patients felt anxious or depressed during measurements (ßanxiety+depression = - 0.26, panxiety+depression < 0.01). CONCLUSIONS: These results do not support the hypothesis that Vitamin D has a positive influence on muscle strength measured by HGS. Nevertheless, this study could confirm the usefulness of HGS as a tool to detect the risk for frequent falls or stumbling. Furthermore, HGS seems to be associated with dizziness and age at onset of menopause. A significant decrease of HGS could also be shown in patients with anxiety and depression. This underlines the importance of interdisciplinary treatment of elderly trauma patients and needs to be taken into account for further studies, as especially the psychological motivation seems to have a significant influence and is sometimes not considered enough in elderly musculo-skeletal patients.

Clinical Acuity in the Emergency Department and Injury Severity Determine Hospital Admission of Older Patients with Low Energy Falls: Outcomes from a Prospective Feasibility Study.

BACKGROUND: Low energy falls (LEF) in older adults constitute a relevant cause for emergency department (ED) visits, hospital admission and in-hospital mortality. Patient-reported outcome measures containing information about patients' medical, mental and social health problems might support disposition and therapy decisions. We investigated the value of a tablet-based (self-)assessment in predicting hospital admission and in-hospital mortality. METHODS: Patients 65 years or older, consecutively presenting with LEF to our level I trauma center ED (from November 2020 to March 2021), were eligible for inclusion in this prospective observational study. The primary endpoint was hospital admission; secondary endpoints were in-hospital mortality and the use of the tablet for self-reported assessment. Multivariate logistic regression models were calculated to measure the association between clinical findings and endpoints. RESULTS: Of 618 eligible patients, 201 patients were included. The median age was 82 years (62.7% women). The hospital admission rate was 45.3% (110/201), with an in-hospital mortality rate of 3.6% (4/110). Polypharmacy (odds ratio (OR): 8.48; 95% confidence interval (95%CI) 1.21-59.37, p = 0.03), lower emergency severity index (ESI) scores (OR: 0.33; 95%CI 0.17-0.64, p = 0.001) and increasing injury severity score (ISS) (OR: 1.54; 95%CI 1.32-1.79, p < 0.001) were associated with hospital admission. The Charlson comorbidity index (CCI) was significantly associated with in-hospital mortality (OR: 2.60; 95%CI: 1.17-5.81, p = 0.03). Increasing age (OR: 0.94; 95%CI: 0.89-0.99, p = 0.03) and frailty (OR: 0.71; 95%CI: 0.51-0.99, p = 0.04) were associated with the incapability of tablet use. CONCLUSIONS: The severity of fall-related injuries and the clinical acuity are easily accessible, relevant predictors for hospital admission. Tablet-based (self-)assessment may be feasible and acceptable during ED visits and might help facilitate comprehensive geriatric assessments during ED stay.

Patients With Dupuytren Disease Use Excessive Grip Force When Lifting and Holding Small Objects, Independent of the Degree of Contracture.

OBJECTIVE: To identify the extent and quality of fine motor skill alteration in patients with Dupuytren disease (DD) using an instrumented device measuring grip forces, beyond the commonly used measurement of contracture. DESIGN: Case-control study. SETTING: University outpatient clinic. PARTICIPANTS: Patients with DD (N=27) and a contracture >45° (Tubiana stage II, III, and IV) were included and compared with age-matched healthy control patients (N=27). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES(S): All individuals were subjected to a set of specific tests using a new instrumented device ("manipulandum"). These included lifting, grasping, and then holding the "manipulandum" with 4 different object characteristics (light and heavy weight, rough and smooth surface) along with a measurement of the precision grip strength. Standard measurements including the Nine-Hole Peg Test, a two-point discrimination, and the Disability of Arm, Shoulder and Hand score were evaluated in comparison. RESULTS: Although the measurements of precision grip, two-point discrimination, Nine-Hole Peg Test, and Disability of Arm, Shoulder and Hand score showed no statistically significant differences between both groups, patients with DD applied significantly greater forces when tested with the different subtests using the "manipulandum." Analysis of the 2-phase movement (lifting and holding the "manipulandum") revealed highly significant differences between the groups. CONCLUSIONS: Patients with DD use excessive grip forces when lifting and holding the "manipulandum" when compared with healthy control patients, independent of the degree of contracture. As no differences in precision grip strength were seen, the presented approach is useful to obtain additional important information about fine motor function in diseased hands.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model.

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells (ADSPCs) in peripheral nerve recovery. Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs. This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned (2% oxygen for 72 hours) autologous ADSPCs (n = 9). This treatment mode was compared with three others: fibrin conduit seeded with ADSPCs cultivated under normoxic conditions (n = 9); non-cell-carrying conduit (n = 9); and nerve autograft only (n = 9). A 16-week follow-up included functional testing (sciatic functional index and static sciatic index) as well as postmortem muscle mass analyses and morphometric nerve evaluations (histology, g-ratio, axon density, and diameter). At 8 weeks, the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups, indicating faster functional regeneration. Furthermore, histologic evaluation showed significantly increased axon outgrowth/branching, axon density, remyelination, and a reduced relative connective tissue area. Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts. Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

Effect of shape and size of supraspinatus tears on rotator cuff strain distribution: an in vitro study.

BACKGROUND: The impact of the size and shape of a supraspinatus tear on the strain of the intact rotator cuff and the kinematics of the shoulder is still unknown. This, however, can be relevant when deciding whether surgical reconstruction is required to prevent an increase in a tendon defect. In this study, the effect of tear width and shape on rotator cuff strain and glenohumeral kinematics was evaluated during active abduction. METHODS: Twelve fresh-frozen cadaveric shoulders with intact rotator cuffs were used in this study. We created 50% and 100% wide (full-thickness) crescent-shaped (CS) tears (n = 6) and reverse L-shaped (rLS) tears (n = 6) in the supraspinatus tendon and measured strain and kinematics during active humeral elevation until 30°. RESULTS: Both tear shapes and sizes led to an increase in internal rotation, supraspinatus loading force, and superior translation of the humerus. For the 100% wide tear size, anterior translation was observed in the CS tear group, whereas in the rLS tear group, this translation occurred mainly in the posterior direction. Strain was higher in the infraspinatus during the first 25° of abduction in comparison with the supraspinatus tendon in both tear shape groups. An analysis of the anterior and posterior tear borders showed a higher strain concentration on the same side of the tear in the CS tear group with 50% and 100% wide tears. CONCLUSIONS: The influence of different tear shapes on translation in the anterior-posterior direction was evident as both CS and rLS tears led to an oppositely directed translation of the humeral head. The strain analysis showed a stress-shielding effect of the infraspinatus at the beginning of abduction. Therefore, special attention must be paid to correctly identify the tear extension and adequately reconstruct the rotator cuff footprint. Moreover, the constant location of maximum strain in the CS tear group may lead to an earlier progression than in the rLS tear group.

Keloids are transcriptionally distinct from normal and hypertrophic scars.

Wound healing and skin regeneration after injury are complex biological processes, and deep injuries with a high degree of tissue destruction may result in severe scar formation. Clinically, scars can be classified into normal, hypertrophic and keloid scars. However, the molecular signature of each scar type is currently not known. The aim of this study was to reveal the transcriptional landscape of normal, hypertrophic and keloid skin scars following hand and plastic surgery based on total RNA sequencing. Eighteen skin scar samples from hand and plastic surgeries of human donors were minced directly after removal and stored in TRIzol (Thermo Fisher, USA). Samples were then subjected to RNA isolation, cDNA library preparation, bulk RNA sequencing and bioinformatics analysis. We show that keloid scars transcriptionally differed from normal and hypertrophic scars. Normal and hypertrophic scars presented overlapping clustering, and eight genes were shown to be commonly expressed between hypertrophic and normal scars. No genes were specifically expressed at a higher level in keloid and normal scars. Based on gene ontology pathway analysis, genes with a higher level of expression in keloid scars lead to increased (extra-) cellular matrix proliferation and cell interaction. Moreover, tumour-like genes were more highly expressed in keloid scars, supporting the clinical impression of strong and diffuse growth. This study furthers our understanding of the classification of differential scar types based on molecular signature, which may shed light on new diagnostic and therapeutic strategies for keloid scars in the future.

Fusion of Normoxic- and Hypoxic-Preconditioned Myoblasts Leads to Increased Hypertrophy.

Injuries, high altitude, and endurance exercise lead to hypoxic conditions in skeletal muscle and sometimes to hypoxia-induced local tissue damage. Thus, regenerative myoblasts/satellite cells are exposed to different levels and durations of partial oxygen pressure depending on the spatial distance from the blood vessels. To date, it is unclear how hypoxia affects myoblasts proliferation, differentiation, and particularly fusion with normoxic myoblasts. To study this, we investigated how 21% and 2% oxygen affects C2C12 myoblast morphology, proliferation, and myogenic differentiation and evaluated the fusion of normoxic- or hypoxic-preconditioned C2C12 cells in 21% or 2% oxygen in vitro. Out data show that the long-term hypoxic culture condition does not affect the proliferation of C2C12 cells but leads to rounder cells and reduced myotube formation when compared with myoblasts exposed to normoxia. However, when normoxic- and hypoxic-preconditioned myoblasts were differentiated together, the resultant myotubes were significantly larger than the control myotubes. Whole transcriptome sequencing analysis revealed several novel candidate genes that are differentially regulated during the differentiation under normoxia and hypoxia in mixed culture conditions and may thus be involved in the increase in myotube size. Taken together, oxygen-dependent adaption and interaction of myoblasts may represent a novel approach for the development of innovative therapeutic targets.

Dental and Orthopaedic Implant Loosening: Overlap in Gene Expression Regulation.

Objectives: Endoprosthetic loosening still plays a major role in orthopaedic and dental surgery and includes various cellular immune processes within peri-implant tissues. Although the dental and orthopaedic processes vary in certain parts, the clinical question arises whether there are common immune regulators of implant loosening. Analyzing the key gene expressions common to both processes reveals the mechanisms of osteoclastogenesis within periprosthetic tissues of orthopaedic and dental origin. Methods: Donor peripheral blood mononuclear cells (PBMCs) and intraoperatively obtained periprosthetic fibroblast-like cells (PPFs) were (co-)cultured with [± macrophage-colony stimulating factor (MCSF) and Receptor Activator of NF-κB ligand (RANKL)] in transwell and monolayer culture systems and examined for osteoclastogenic regulations [MCSF, RANKL, osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)] as well as the ability of bone resorption. Sequencing analysis compared dental and orthopaedic (co-)cultures. Results: Monolayer co-cultures of both origins expressed high levels of OPG, resulting in inhibition of osteolysis shown by resorption assay on dentin. The high OPG-expression, low RANKL/OPG ratios and a resulting inhibition of osteolysis were displayed by dental and orthopaedic PPFs in monolayer even in the presence of MCSF and RANKL, acting as osteoprotective and immunoregulatory cells. The osteoprotective function was only observed in monolayer cultures of dental and orthopaedic periprosthetic cells and downregulated in the transwell system. In transwell co-cultures of PBMCs/PPFs profound changes of gene expression, with a significant decrease of OPG (20-fold dental versus 100 fold orthopaedic), were identified. Within transwell cultures, which offer more in vivo like conditions, RANKL/OPG ratios displayed similar high levels to the original periprosthetic tissue. For dental and orthopaedic implant loosening, overlapping findings in principal component and heatmap analysis were identified. Conclusions: Thus, periprosthetic osteoclastogenesis may be a correlating immune process in orthopaedic and dental implant failure leading to comparable reactions with regard to osteoclast formation. The transwell cultures system may provide an in vivo like model for the exploration of orthopaedic and dental implant loosening.

Correction to: Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

An amendment to this paper has been published and can be accessed via the original article.

Bone defect reconstruction with a novel biomaterial containing calcium phosphate and aluminum oxide reinforcement.

BACKGROUND: Reconstruction of metaphyseal fractures represents a clinical challenge for orthopedic surgeons. Especially in osteoporotic bone, these fractures are frequently accompanied by osseous substance defects. In order to ensure rapid mobilization of patients, high stability requirements must be met by osteosynthesis. Various bone graft materials have been introduced in the past, such as autologous bone or exogenous bone substitute materials. These are used as bone void fillers or as augmentation techniques to ensure safe fixation of osteosynthesis. New calcium phosphate-based bone void-filling materials could be a promising alternative to autologous bone or to the currently and widely used polymethylmethacrylate (PMMA)-based cement. The aim of this study was to evaluate a novel paste-like bone void filler in vivo and in vitro with regard to biocompatibility and osteoconductivity. METHODS: In addition to in vitro testing of cell compatibility using pre-osteoblasts (MC3T3-E1), 35 Wistar rats were treated in vivo with implantation of various material mixtures based on calcium phosphate and aluminum oxide reinforcement in a metaphyseal drill hole defect. After 4 weeks, an examination by micro-computed tomography (µCT) and histology was performed. RESULTS: The in vitro analysis showed good biocompatibility with a high cell survival of osteoblasts. In the in vivo experiments, a significantly higher bone ingrowth compared to the empty defect was shown by µCT and histological analysis. Here, the group receiving material reinforced with aluminum oxide (Al2O3) showed a bone volume/tissue volume (BV/TV) of 89.19% compared to a BV/TV of 83.14% for the empty defect (p = 0.0013). In the group treated with a polysaccharide matrix, no increase in BV/TV was observed given a mean ratio of 80.14%. Scoring of histological sections did not reveal a significant difference between CaP and CaP that was substituted with Al2O3. CONCLUSION: The results of this study show an encouraging first step towards the development of new pasty, bone void-filling materials. We demonstrated that a new paste-like bone-filling material, based on calcium phosphate granulates and aluminum oxide to provide strength, exhibits good biocompatibility and osteoconductivity. Further biomechanical test in an osteoporotic animal model will have to be performed, to prove feasibility in metaphyseal defects.

How We Manage Bone Marrow Edema-An Interdisciplinary Approach.

Bone marrow edema (BME) is a descriptive term for a common finding in magnetic resonance imaging (MRI). Although pain is the major symptom, BME differs in terms of its causal mechanisms, underlying disease, as well as treatment and prognosis. This complexity together with the lack of evidence-based guidelines, frequently makes the identification of underlying conditions and its management a major challenge. Unnecessary multiple consultations and delays in diagnosis as well as therapy indicate a need for interdisciplinary clinical recommendations. Therefore, an interdisciplinary task force was set up within our large osteology center consisting of specialists from internal medicine, endocrinology/diabetology, hematology/oncology, orthopedics, pediatrics, physical medicine, radiology, rheumatology, and trauma surgery to develop a consenus paper. After review of literature, review of practical experiences (expert opinion), and determination of consensus findings, an overview and an algorithm were developed with concise summaries of relevant aspects of the respective underlying disease including diagnostic measures, clinical features, differential diagnosis and treatment of BME. Together, our single-center consensus review on the management of BME may help improve the quality of care for these patients.

Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model.

BACKGROUND: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible. METHODS: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 µl saline, 100 µl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 µl hydrogel loaded with 1 µg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination. RESULTS: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples. CONCLUSIONS: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.

Sarcopenia - Endocrinological and Neurological Aspects.

Sarcopenia in geriatric patients is often associated with or even caused by changes of the endocrine and nervous system. The multifactorial pathogenesis of sarcopenia and additional multimorbidity in geriatric patients makes it difficult to study distinct pathogenic pathways leading to sarcopenia. Patients suffering from diabetes, Cushing's syndrome, chronic kidney disease, Klinefelter's syndrome or motor neuron diseases, such as amyotrophic lateral sclerosis for example are known to have impaired muscle property and reduced physical performance. These patients are typically younger and suffer from conditions caused by a known molecular disease mechanism and a peculiar sarcopenic phenotype. Therefore, these sequelae can serve as prototypic disease models to study isolated endocrinological and neurodegenerative causes for sarcopenia. This review focuses on diseases whose etiopathogenesis of muscle impairment is known. The idea is to use these diseases as proof of principles to develop a classification algorithm of sarcopenia in the elderly to make a more mechanism-oriented therapy be possible.

Validation of a novel animal model for sciatic nerve repair with an adipose-derived stem cell loaded fibrin conduit.

Despite the regenerative capabilities of peripheral nerves, severe injuries or neuronal trauma of critical size impose immense hurdles for proper restoration of neuro-muscular circuitry. Autologous nerve grafts improve re-establishment of connectivity, but also comprise substantial donor site morbidity. We developed a rat model which allows the testing of different cell applications, i.e., mesenchymal stem cells, to improve nerve regeneration in vivo. To mimic inaccurate alignment of autologous nerve grafts with the injured nerve, a 20 mm portion of the sciatic nerve was excised, and sutured back in place in reversed direction. To validate the feasibility of our novel model, a fibrin gel conduit containing autologous undifferentiated adipose-derived stem cells was applied around the coaptation sites and compared to autologous nerve grafts. After evaluating sciatic nerve function for 16 weeks postoperatively, animals were sacrificed, and gastrocnemius muscle weight was determined along with morphological parameters (g-ratio, axon density & diameter) of regenerating axons. Interestingly, the addition of undifferentiated adipose-derived stem cells resulted in a significantly improved re-myelination, axon ingrowth and functional outcome, when compared to animals without a cell seeded conduit. The presented model thus displays several intriguing features: it imitates a certain mismatch in size, distribution and orientation of axons within the nerve coaptation site. The fibrin conduit itself allows for an easy application of cells and, as a true critical-size defect model, any observed improvement relates directly to the performed intervention. Since fibrin and adipose-derived stem cells have been approved for human applications, the technique can theoretically be performed on humans. Thus, we suggest that the model is a powerful tool to investigate cell mediated assistance of peripheral nerve regeneration.

Comparison of different strategies for in vivo seeding of prevascularized scaffolds.

Scaffolds seeded with multipotent precursor cells were hypothesized to heal critically sized bone defects. However, the success of this concept was limited by low cell survival after transplantation due to a lack of nutrients and oxygen. In vivo prevascularization of scaffolds before cell seeding may improve cell survival, yet the best seeding technique and time point of cell application remain elusive. Thus, the aim of this study was to compare different strategies. Demineralized bone matrix scaffolds were implanted around the saphenous arteriovenous (AV) bundle in nude mice. In vivo seeding was performed 0, 5, or 21 days after implantation using enhanced green fluorescent protein (eGFP)-expressing mesenchymal stem cells (MSCs). Cells were applied either by injection or the repetitive dripping technique. In vitro seeded and subcutaneously implanted scaffolds served as controls. Fourteen days after cell application, the fluorescence intensity of transplanted cells and the extent of newly formed vessels were quantified. We found that the AV flow through model as well as cell application increased vessel formation. In vitro seeding resulted in significantly higher cell numbers than in vivo seeding. With increasing time of prevascularization, the number of cells declined dramatically. In vivo seeding by cell injection was superior to the repetitive dripping protocol. On subcutaneously implanted scaffolds, significantly, more cells were found than on axially perfused scaffolds. We conclude that in vitro seeding is more efficient compared to the two novel in vivo seeding techniques of prevascularized scaffolds. With increasing time of prevascularization, the seeding efficiency for the in vivo methods further decreases, presumably due to the ingrowth of connective tissue. Even though, the presence of MSCs and the longer period of prevascularization enhances vessel formation, this conceivable advantage is limited supposedly by the inferior seeding efficiency.

Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy.

The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.