Advancements in cell-based therapies for the treatment of pressure injuries: A systematic review of interventional studies.

The high recurrence and complications associated with severe pressure injuries (PI) necessitate the exploration of advanced treatments, such as cell-based therapies, to facilitate wound healing. Such techniques harness the ability of different cell types to promote angiogenesis, re-epithelialization of the skin, and tissue regeneration. This systematic review explores the efficacy of cell-based therapies and tissue engineering in treating deep PI. We searched for interventional studies using cells in the treatment of PI in adults in four online libraries (PubMed, Embase, Ovid Medline, and Cochrane; latest search 10th June 2023). We found one randomized clinical trial (RCT), two non-RCT, and three pre-post studies, comprising 481 study participants with PI (253 intervention/228 controls). The risk of bias was categorized as moderate due to minimal bias in outcome measurements, or high owing to unclear patient randomization methods, as assessed by the ROBINS-I, NIH, and RoB-2 tools. Four cell types were identified in the context of cell-based therapies of PI: bone marrow mononuclear stem cells (BM-MNCs, n = 2); hematopoietic derived stem cells (HSC, n = 1); macrophages and activated macrophage suspensions (AMS, n = 2); and cryopreserved placental membrane containing viable cells (vCPM, n = 1). Wound healing outcomes were observed in patients undergoing cell-based therapies, including complete wound closure (AMS, vCPM; n = 142), faster healing rate (BM-MNCs, AMS; n = 146), improved granulation tissue formation (HSC, n = 3) and shorter hospitalization time (BM-MNCs; n = 108) compared to standard of care, with no adverse reactions. PI healing rate decreased only in one study with BM-MNC therapy, compared to control (n = 86). Based on the available data, though with limited evidence, it seems that macrophage deployment showed the most favorable outcomes. The results indicate that cell-based therapies offer a potential avenue for enhancing wound healing and tissue repair in PI; however, more extensive research is needed in this domain.

Understanding the impact of spinal cord injury on the microbiota of healthy skin and pressure injuries.

Pressure injuries (PI) are a common issue among individuals with spinal cord injury (SCI), especially in the sitting areas of the body. Considering the risk of infections occurring to PI during the wound healing process, the skin microbiome is likely to be a source of bacteria. We investigated the relationship between skin and PI microbiomes, and assessed any correlation with clinically relevant outcomes related to PI. Samples were isolated from SCI patients undergoing reconstructive surgery of PI, severity grades III and IV. DNA samples from skin and PI were analysed using 16S rRNA gene sequencing. Our results showed disparities in microbiome composition between skin and PI. The skin had lower diversity, while PI showed increased bacterial homogeneity as the severity grade progressed. The skin bacterial composition varied based on its location, influenced by Cutibacterium. Compositional differences were identified between PI grades III and IV, with clusters of bacteria colonizing PI, characterized by Pseudomonas, Proteus and Peptoniphilus. The skin and PI microbiomes were not affected by the level of the SCI. Our study highlights the differences in the microbiome of skin and PI in SCI patients. These findings could be used to target specific bacteria for PI treatment in clinical practice.

Assessing the Feasibility of a Multimodal Approach to Pain Evaluation in Early Stages after Spinal Cord Injury.

This research evaluates the feasibility of a multimodal pain assessment protocol during rehabilitation following spinal cord injury (SCI). The protocol amalgamates clinical workup (CW), quantitative sensory testing (QST), and psychosocial factors (PSF) administered at 4 (T1), 12 (T2), and 24 (T3) weeks post injury and at discharge (T4). Molecular blood biomarkers (BB) were evaluated via gene expression and proteomic assays at T1 and T4. Different pain trajectories and temporal changes were identified using QST, with inflammation and pain-related biomarkers recorded. Higher concentrations of osteopontin and cystatin-C were found in SCI patients compared to healthy controls, indicating their potential as biomarkers. We observed altered inflammatory responses and a slight increase in ICAM-1 and CCL3 were noted, pointing towards changes in cellular adhesion linked with spinal injury and a possible connection with neuropathic pain. Despite a small patient sample hindering the correlation of feasibility data, descriptive statistical analyses were conducted on stress, depression, anxiety, quality of life, and pain interferences. The SCI Pain Instrument (SCIPI) was efficient in distinguishing between nociceptive and neuropathic pain, showing a progressive increase in severity over time. The findings emphasize the need for the careful consideration of recruitment setting and protocol adjustments to enhance the feasibility of multimodal pain evaluation studies post SCI. They also shed light on potential early adaptive mechanisms in SCI pathophysiology, warranting the further exploration of prognostic and preventive strategies for chronic pain in the SCI population.

Endocrinological and inflammatory markers in individuals with spinal cord injury: A systematic review and meta-analysis.

Spinal cord injury (SCI) can lead to dramatic physiological changes which can be a factor in developing secondary health conditions and might be reflected in biomarker changes in this elevated risk group. We focused specifically on the endocrine and inflammation profile differences between SCI and able-bodied individuals (ABI). Our aim was to determine the differences in inflammatory markers and endocrine profiles between SCI and ABI. We systematically searched 4 electronic databases for relevant studies. Human observational (cross-sectional, cohort, case-control) studies that compared biomarkers of interest between SCI and ABI population were included. Weighted mean difference between SCI and ABI was calculated using random-effects models. Heterogeneity was computed using I2 statistic and chi-squared test. Study quality was evaluated through the Newcastle-Ottawa Scale. The search strategy yielded a total of 2,603 studies from which 256 articles were selected for full-text assessment. Sixty-two studies were included in the meta-analysis. SCI individuals had higher levels of pro-inflammatory C-reactive protein and IL-6 than ABI. Creatinine and 25-hydroxyvitamin D3 levels were lower in SCI than ABI. Total testosterone levels and IGF-1 were also found to be lower, while cortisol and leptin levels were higher in SCI when compared to ABI. Accordingly, meta-regression, subgroup analysis, and leave-one-out analysis were performed, however, they were only able to partially explain the high levels of heterogeneity. Individuals with SCI show higher levels of inflammatory markers and present significant endocrinological changes when compared to ABI. Moreover, higher incidence of obesity, diabetes, osteoporosis, and hypogonadism in SCI individuals, together with decreased creatinine levels reflect some of the readily measurable aspects of the phenotype changes in the SCI group. These findings need to be considered in anticipating medically related complications and personalizing SCI medical care.

Physical activity and cardiometabolic risk factors in individuals with spinal cord injury: a systematic review and meta-analysis.

Physical inactivity in individuals with spinal cord injury (SCI) has been suggested to be an important determinant of increased cardiometabolic disease (CMD) risk. However, it remains unclear whether physically active SCI individuals as compared to inactive or less active individuals have truly better cardiometabolic risk profile. We aimed to systematically review and quantify the association between engagement in regular physical activity and/or exercise interventions and CMD risk factors in individuals with SCI. Four medical databases were searched and studies were included if they were clinical trials or observational studies conducted in adult individuals with SCI and provided information of interest. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was applied to rate the certainty of evidence. Of 5816 unique citations, 11 randomized clinical trials, 3 non-randomized trial and 32 cross-sectional studies comprising more than 5500 SCI individuals were included in the systematic review. In meta-analysis of RCTs and based on evidence of moderate certainty, physical activity in comparison to control intervention was associated with: (i) better glucose homeostasis profile [WMD of glucose, insulin and Assessment of Insulin Resistance (HOMA-IR) were - 3.26 mg/dl (95% CI - 5.12 to - 1.39), - 3.19 µU/ml (95% CI - 3.96 to - 2.43)] and - 0.47 (95% CI - 0.60 to - 0.35), respectively], and (ii) improved cardiorespiratory fitness [WMD of relative and absolute oxygen uptake relative (VO2) were 4.53 ml/kg/min (95% CI 3.11, 5.96) and 0.26 L/min (95% CI 0.21, 0.32) respectively]. No differences were observed in blood pressure, heart rate and lipids (based on evidence of low/moderate certainty). In meta-analysis of cross-sectional studies and based on the evidence of very low to low certainty, glucose [WMD - 3.25 mg/dl (95% CI - 5.36, - 1.14)], insulin [- 2.12 µU/ml (95% CI - 4.21 to - 0.03)] and total cholesterol [WMD - 6.72 mg/dl (95% CI - 13.09, - 0.34)] were lower and HDL [WMD 3.86 mg/dl (95% CI 0.66, 7.05)] and catalase [0.07 UgHb-1 (95% CI 0.03, 0.11)] were higher in physically active SCI individuals in comparison to reference groups. Based on limited number of cross-sectional studies, better parameters of systolic and diastolic cardiac function and lower carotid intima media thickness were found in physically active groups. Methodologically sound clinical trials and prospective observational studies are required to further elaborate the impact of different physical activity prescriptions alone or in combination with other life-style interventions on CMD risk factors in SCI individuals.

Systematic Review of Human and Animal Evidence on the Role of Buckwheat Consumption on Gastrointestinal Health.

BACKGROUND: Buckwheat is a commonly cultivated crop with growing evidence that it is beneficial to gastrointestinal (GI) health. This systematic review summarizes the role of buckwheat in modifying GI health outcomes and microbiomes. METHODS: Four medical databases and Google Scholar were systematically searched. Clinical trials, observational studies, animal in vivo, and in vitro studies with human and animal GI-derived samples were included. RESULTS: There were 32 studies (one randomized controlled trial [RCT], one non-randomized trial, 3 observational, 9 in vitro, and 18 animal in vivo studies) included. In preclinical studies, buckwheat extracts were observed to have cytotoxic potential against human-derived GI cancer cell lines. Animals fed with buckwheat had lower GI mucosal inflammation, higher alpha diversity in the GI microbiome, and higher levels of fecal short-chain fatty acids. Human evidence studies and clinical trials were limited and predominantly of moderate risk of bias. The majority of in vitro studies with GI-derived samples and in vivo studies were reliable without restrictions in study design. CONCLUSION: In vivo and in vitro studies show that buckwheat may have potential GI benefits due to its anti-oxidant and anti-inflammatory potential; however, human evidence remains limited, and its impact on health in humans remains to be elucidated in future trials.

Virtual and Augmented Reality Applications in Medicine: Analysis of the Scientific Literature.

BACKGROUND: Virtual reality (VR) and augmented reality (AR) have recently become popular research themes. However, there are no published bibliometric reports that have analyzed the corresponding scientific literature in relation to the application of these technologies in medicine. OBJECTIVE: We used a bibliometric approach to identify and analyze the scientific literature on VR and AR research in medicine, revealing the popular research topics, key authors, scientific institutions, countries, and journals. We further aimed to capture and describe the themes and medical conditions most commonly investigated by VR and AR research. METHODS: The Web of Science electronic database was searched to identify relevant papers on VR research in medicine. Basic publication and citation data were acquired using the "Analyze" and "Create Citation Report" functions of the database. Complete bibliographic data were exported to VOSviewer and Bibliometrix, dedicated bibliometric software packages, for further analyses. Visualization maps were generated to illustrate the recurring keywords and words mentioned in the titles and abstracts. RESULTS: The analysis was based on data from 8399 papers. Major research themes were diagnostic and surgical procedures, as well as rehabilitation. Commonly studied medical conditions were pain, stroke, anxiety, depression, fear, cancer, and neurodegenerative disorders. Overall, contributions to the literature were globally distributed with heaviest contributions from the United States and United Kingdom. Studies from more clinically related research areas such as surgery, psychology, neurosciences, and rehabilitation had higher average numbers of citations than studies from computer sciences and engineering. CONCLUSIONS: The conducted bibliometric analysis unequivocally reveals the versatile emerging applications of VR and AR in medicine. With the further maturation of the technology and improved accessibility in countries where VR and AR research is strong, we expect it to have a marked impact on clinical practice and in the life of patients.

Autofluorescence-based sorting removes senescent cells from mesenchymal stromal cell cultures.

Mesenchymal stromal cells (MSC) are used in cell therapy, but results depend on the unknown quality of cell populations. Extended culture time of MSC increases their senescent levels, leading to a critical loss of cell fitness. Here, we tested the suitability of MSC-sorting based on their FACS autofluorescence profile, for a rapid and non-invasive method of senescent cell elimination. Cells were classified in low- (LA) and high- (HA) autofluorescence groups, and results compared to the original MSC population (control). Three days after sorting, cells were screened by replicative senescence markers (cell volume, SA-ß-Gal assay and gene/protein expression) and MSC differentiation assays. The transcriptional profiles of sorted MSC were also analyzed by RNA-Seq. Compared to control, LA cells had 10% lower cell volume and autofluorescence, and 50% less SA-ß-Gal + cells. Instead, HA cells had 20% higher cell volume and autofluorescence, and 120% more SA-ß-Gal + cells. No changes in replicative senescence and differentiation potentials were observed between all groups. However, 68 genes (16 related to senescence) were significantly differentially expressed (DEG) between LA and other groups. Biological network of DEG identified CXCL12 as topological bottleneck. In summary, MSC sorting may have practical clinical implications to enhance the results of MSC-based therapies.

Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies' outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-ß-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-ß-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.

Treatment of Naturally Degenerated Canine Lumbosacral Intervertebral Discs with Autologous Mesenchymal Stromal Cells and Collagen Microcarriers: A Prospective Clinical Study.

Intervertebral disc (IVD) degeneration is a frequent disease in modern societies and at its later stages is likely to cause chronic low back pain. Although many studies have been published, the available treatments for IVD degeneration fail to promote regeneration or even marginal repair of the IVD structure. In this study, we aimed to establish veterinary canine patients as a translational large animal model that recapitulates IVD degeneration that occurs in humans, and to investigate the suitability of intradiscal application of mesenchymal stromal cells (MSC). Twenty client-owned dogs diagnosed with spontaneous degenerative lumbosacral IVD and low back pain were included in the study. Autologous MSC were isolated from bone marrow and cultured for 2 weeks. Prior to injection, MSC were attached on collagen microcarriers for delivery, with or without TGF-ß1 crosslinking. After decompressive spinal surgery, dogs received an intradiscal injection of MSC-microcarriers ( n = 11), MSC-TGF-ß1-microcarriers ( n = 6) or microcarriers only (control, n = 3). MSC-microcarriers were initially evaluated in vitro and ex vivo, to test cell chondrogenic potential and biomechanical properties of the microcarriers, respectively. Clinical performance and Pfirrmann grading were evaluated at 10 months after the injection by magnetic resonance imaging. MSC differentiated successfully in vitro towards chondrogenic phenotype and biomechanical tests showed no significant differences of IVD stiffness after microcarrier injection. In vivo injection was successful in all dogs, without any visible leakage, and clinical functioning was restored back to normality. However, postoperative Pfirrmann grade remained identical in all dogs, and formation of Schmorl's nodes was detected in 45% of dogs. This side effect was reduced by halving the injection volume, which was then observed only in 11% of dogs. In conclusion, we observed marked clinical improvement in all groups, despite the formation of Schmorl's nodes, but microcarriers and MSC failed to regenerate the structure of degenerated IVD.

Unexpectedly decreased plasma cytokines in patients with chronic back pain.

INTRODUCTION: Chronic back pain is one of the most important socioeconomic problems that affects the global population. Elevated levels of inflammatory mediators, such as cytokines, have been correlated with pain, but their role in chronic back pain remains unclear. The effectiveness of anti-inflammatory drugs seems to be limited for chronic back pain. The authors wanted to investigate the levels of inflammatory mediators in long-term medically treated patients with persistent chronic back pain. METHODS: Cytokine plasma levels of patients with chronic back pain (n=23), compared to pain-free healthy controls (n=30), were investigated by immunoassay. Patients with chronic back pain were exposed to long-term conservative medical therapy with physiotherapy and anti-inflammatories, also combined with antidepressants and/or muscle-relaxants. RESULTS: The patients with chronic back pain expressed lower levels of the chemokines MCP1, CCL5, and CXCL6 compared to pain-free healthy controls. Significantly lower concentrations of the anti-inflammatory cytokines, interleukin (IL)-4 and granulocyte-colony stimulating factor were also found. Interestingly, levels of proinflammatory cytokines (IL-2, IL-6, IL-1ß, tumor necrosis factor alpha), IL-10, granulocyte-macrophage colony-stimulating factor, and stromal cell-derived factor 1 alpha showed no significant differences between both groups. CONCLUSION: This decrease of inflammatory mediators in medically treated patients with chronic back pain is of unclear origin and might be either a long-term side effect of medical therapy or related to chronic pain. Further longitudinal research is necessary to elucidate the underlying cause of these findings.

VEGF vascularization pathway in human intervertebral disc does not change during the disc degeneration process.

OBJECTIVE: During degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration. RESULTS: Expression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of the classic vascularization pathway is stable across degeneration stages and we assume that soluble vascular endothelial growth factor receptor 1 does not contribute to prevent disc degeneration. However, we observed increased expression levels of genes involved in alternative vascularization signalling pathways in severely degenerated discs, suggesting that abnormal vascularization is part of the pathological progression of disc degeneration.

Bone Marrow-Derived Mesenchymal Stem Cells as Autologous Therapy in Dogs with Naturally Occurring Intervertebral Disc Disease: Feasibility, Safety, and Preliminary Results.

The intradiscal application of mesenchymal stem cells (MSCs) is considered a promising strategy for intervertebral disc (IVD) regeneration. Although many studies have been published, the feasibility and regenerative effects of intradiscal MSC application have not been evaluated in an animal model suffering from naturally occurring degenerative disc disease. Six German Shepherd Dogs suffering from naturally occurring degenerative IVD disease were included. Autologous MSCs were isolated from bone marrow (iliac crest) and cultured for 3 weeks. After decompressive spinal surgery, three dogs received an intradiscal injection of MSCs, while the other three dogs received an intradiscal injection of saline (control). Clinical status, disc height index, Pfirrmann grading, and disc volumetry were evaluated at 1, 6, and 12 months after treatment. Autologous application of canine MSCs was feasible and successful in all dogs. No evident complication was found. Surgery resulted in an equal improvement in clinical status in the treatment and control dogs. In the MSC group, the Pfirrmann grade increased in all patients over time, whereas in the control group, the Pfirrmann grade remained stable. The volume of the L7-S1 IVD gradually increased during the 12-month study period in all dogs, with no evident difference between the MSC and control group. On the basis of this preliminary study, it can be concluded that intradiscal injection of autologous MSCs in dogs with spontaneous degenerative IVD disease is well tolerated without any adverse effects, does not affect clinical outcome, and does not have any evident regenerative effects.

Oxidative status predicts quality in human mesenchymal stem cells.

BACKGROUND: Human bone marrow-derived mesenchymal stem cells (MSC) are adult progenitor cells with great potential for application in cell-based therapies. From a cell-based therapy perspective, there are two limitations to MSC use: (1) these therapies require large numbers of cells, and long-term expansion of MSC in vitro promotes replicative senescence; and (2) patient variability is a challenge for defining MSC quality standards for transplantation. This study aimed to determine whether low or high oxidative status of MSC correlate with changes in cell expansion and differentiation potentials. METHODS: We investigated functional aspects of mitochondria, such as cell metabolic activity indicators and expression of antioxidant enzymes. Furthermore, we tested if senescence-induced changes in oxidative status of MSC could be counteracted by methylene blue (MB), an alternative mitochondrial electron transfer known to enhance cell bioenergetics. RESULTS: MSC isolated from donors of the same age showed distinctive behavior in culture and were grouped as weak (low colony-forming units (CFU) and a short life in vitro) and vigorous MSC (high CFU and a long life in vitro). In comparison to weak MSC, vigorous MSC had oxidative status characterized by lower mitochondrial membrane potential, lower mitochondrial activity, and fewer reactive oxygen species production, as well as reduced mitochondrial biogenesis. Vigorous MSC had a significantly higher expansion potential compared to weak MSC, while no differences were observed during differentiation. MB treatment significantly improved expansion and differentiation potential, however only in vigorous MSC. CONCLUSIONS: Together, these results demonstrate the importance of mitochondrial function in MSC in vitro, and that cells with low oxidative status levels are better candidates for cell-based therapies.

Increased motility of mesenchymal stem cells is correlated with inhibition of stimulated peripheral blood mononuclear cells in vitro.

Immunomodulatory properties of mesenchymal stem cells (MSC) are key components of their successful applications in clinical setting. However, treatments based on MSC immunomodulation need understanding of cell characteristics before cell transplantation. We used live-imaging to test the suitability of the MSC motility as a parameter for quick prediction of the immunomodulatory potential of human MSC in regulating the activity of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Bone marrow MSC, from various donors and in vitro passages, were cultured with or without stimulated PBMC. After seven days, immunomodulation was assessed by measuring PBMC proliferation, IgG production and cytokine secretion in MSC and PBMC monocultures and co-cultures, and results were correlated to MSC motility. In co-culture, we observed that MSC successfully inhibited PBMC activity, reducing PBMC proliferation and IgG production compared to PBMC monoculture. MSC modulated PBMC to reduce the secretion of TNFα and IL-10, increase IL-6, G-CSF and MCP-1, while GM-CSF was not affected. By live-imaging tracking of cell trajectories, we observed that fast moving MSC were inhibiting more efficiently stimulated PBMC compared to slow ones. In co-culture, fast MSC were more effective in inhibiting IgG production (˜30% less IgG), and secreted higher levels of IL-10 (˜10% increase) and GM-CSF (˜20% increase) compared to slower cells. Furthermore, fast MSC in monocultures produced 2.3-fold more IL-6, 1.5-fold MCP-1 and 1.2-fold G-CSF in comparison to slower cells. In conclusion, live-imaging cell tracking allowed us to develop an indicative assay of the immune-regulatory potential of MSC prior to in vivo administration. Key Words: Human mesenchymal stem cells, Immunomodulatory potential, In vitro cell motility, Stem cell transplantation.

An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated ß-galactosidase (SA-ß-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-ß-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Tissue engineering is a field in progressive expansion and requires constant updates in methods and devices. One of the central fields is the development of biocompatible, biodegradable, and injectable scaffolds, such as collagen microcarriers. To enhance cell attachment and produce a cost-effective cell culture solution with local stimulation of cells, basic fibroblast growth factor (bFGF) or transforming growth factor-ß1 (TGF-ß1) was covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with bFGF or TGF-ß1 were used for expansion and chondrogenic differentiation of human mesenchymal stem cells (MSCs). Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. MSCs effectively proliferated on microcarriers cross-linked with bFGF, especially by EDC/NHS cross-linking. Chondrogenic differentiation of MSCs was induced by TGF-ß1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. In conclusion, cross-linking of bFGF or TGF-ß1 to collagen microcarriers supported in vitro proliferation and chondrogenesis, respectively. If translated in vivo and in clinical practice, such approach might lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.

An enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug - clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

In vitro cell motility as a potential mesenchymal stem cell marker for multipotency.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.

Canine Mesenchymal Stem Cell Potential and the Importance of Dog Breed: Implication for Cell-Based Therapies.

The study of canine bone marrow-derived mesenchymal stem cells (MSCs) has a prominent position in veterinary cell-based applications. Yet the plethora of breeds, their different life spans, and interbreed variations provide unclearness on what can be achieved specifically by such therapies. In this study, we compared a set of morphological, physiological, and genetic markers of MSCs derived from large dog breeds, namely, Border collie, German shepherd, Labrador, Malinois, Golden retriever, and Hovawart. We compared colony-forming units (CFUs) assay, population doubling time (PDT), senescence-associated ß-galactosidase (SA-ß-gal) activity, telomere length, and gene expression of MSCs, as well as the ability of cells to differentiate to osteogenic, adipogenic, and chondrogenic phenotypes. The influence of the culture media α-MEM, low-glucose DMEM, and high-glucose DMEM, used in cell isolation and expansion, was investigated in the presence and absence of basic fibroblast growth factor (bFGF). Initial cell yield was not affected by culturing medium, but MSCs expanded best in α-MEM supplemented with bFGF. After isolation, the number of MSCs was similar among breeds--as shown by equivalent CFUs--except in the Hovawart samples, which had fivefold less CFU. Telomere lengths were similar among breeds. MSCs divided actively only for 4 weeks in culture (PDT = ∼50 h/division), except Border collie cells divided for a longer time than cells from other groups. The percentage of senescent cells increased linearly in all breeds with time, with a faster rate in German shepherd, Labrador, and Golden retriever. Border collie cells underwent efficient osteogenic differentiation, Hovawart cells performed the best in chondrogenic differentiation, and Labrador cells in both, while German shepherd cells had the lower differentiation potential. MSCs from all breeds preserved the same adipogenic differentiation potential. In conclusion, despite variations, isolated MSCs can be expanded and differentiated in vitro, and all breeds are eligible for MSC-based therapies.