Supporting active engagement of adults with intellectual disabilities in lifestyle modification interventions: a realist evidence synthesis of what works, for whom, in what context and why.

BACKGROUND: Lifestyle modification interventions for adults with intellectual disabilities have had, to date, mixed effectiveness. This study aimed to understand how lifestyle modification interventions for adults with intellectual disabilities work, for whom they work and in what circumstances. METHODS: A realist evidence synthesis was conducted that incorporated input from adults with intellectual disabilities and expert researchers. Following the development of an initial programme theory based on key literature and input from people with lived experience and academics working in this field, five major databases (MEDLINE, EMBASE, CINAHL, PsycINFO and ASSIA) and clinical trial repositories were systematically searched. Data from 79 studies were synthesised to develop context, mechanism and outcome configurations (CMOCs). RESULTS: The contexts and mechanisms identified related to the ability of adults with intellectual disabilities to actively take part in the intervention, which in turn contributes to what works, for whom and in what circumstances. The included CMOCs related to support involvement, negotiating the balance between autonomy and behaviour change, fostering social connectedness and fun, accessibility and suitability of intervention strategies and delivery and broader behavioural pathways to lifestyle change. It is also essential to work with people with lived experiences when developing and evaluating interventions. CONCLUSIONS: Future lifestyle interventions research should be participatory in nature, and accessible data collection methods should also be explored as a way of including people with severe and profound intellectual disabilities in research. More emphasis should be given to the broader benefits of lifestyle change, such as opportunities for social interaction and connectedness.

Lifestyle modification interventions for adults with intellectual disabilities: systematic review and meta-analysis at intervention and component levels.

BACKGROUND: Adults with intellectual disabilities (IDs) are susceptible to multiple health risk behaviours such as alcohol consumption, smoking, low physical activity, sedentary behaviour and poor diet. Lifestyle modification interventions can prevent or reduce negative health consequences caused by these behaviours. We aim to determine the effectiveness of lifestyle modification interventions and their components in targeting health risk behaviours in adults with IDs. METHODS: A systematic review and meta-analysis were conducted. Electronic databases, clinical trial registries, grey literature and citations of systematic reviews and included studies were searched in January 2021 (updated February 2022). Randomised controlled trials and non-randomised controlled trials targeting alcohol consumption, smoking, low physical activity, sedentary behaviours and poor diet in adults (aged ≥ 18 years) with ID were included. Meta-analysis was conducted at the intervention level (pairwise and network meta-analysis) and the component-level (component network meta-analysis). Studies were coded using Michie's 19-item theory coding scheme and 94-item behaviour change taxonomies. Risk of bias was assessed using the Cochrane Risk of Bias (ROB) Version 2 and Risk of Bias in Non-randomised Studies of Interventions (ROBINS-I). The study involved a patient and public involvement (PPI) group, including people with lived experience, who contributed extensively by shaping the methodology, providing valuable insights in interpreting results and organising of dissemination events. RESULTS: Our literature search identified 12 180 articles, of which 80 studies with 4805 participants were included in the review. The complexity of lifestyle modification intervention was dismantled by identifying six core components that influenced outcomes. Interventions targeting single or multiple health risk behaviours could have a single or combination of multiple core-components. Interventions (2 RCTS; 4 non-RCTs; 228 participants) targeting alcohol consumption and smoking behaviour were effective but based on limited evidence. Similarly, interventions targeting low physical activity only (16 RCTs; 17 non-RCTs; 1413 participants) or multiple behaviours (low physical activity only, sedentary behaviours and poor diet) (17 RCTs; 24 non-RCTs; 3164 participants) yielded mixed effectiveness in outcomes. Most interventions targeting low physical activity only or multiple behaviours generated positive effects on various outcomes while some interventions led to no change or worsened outcomes, which could be attributed to the presence of a single core-component or a combination of similar core components in interventions. The intervention-level meta-analysis for weight management outcomes showed that none of the interventions were associated with a statistically significant change in outcomes when compared with treatment-as-usual and each other. Interventions with core-components combination of energy deficit diet, aerobic exercise and behaviour change techniques showed the highest weight loss [mean difference (MD) = -3.61, 95% credible interval (CrI) -9.68 to 1.95] and those with core-components combination dietary advice and aerobic exercise showed a weight gain (MD 0.94, 95% CrI -3.93 to 4.91). Similar findings were found with the component network meta-analysis for which additional components were identified. Most studies had a high and moderate risk of bias. Various theories and behaviour change techniques were used in intervention development and adaptation. CONCLUSION: Our systematic review is the first to comprehensively explore lifestyle modification interventions targeting a range of single and multiple health risk behaviours in adults with ID, co-produced with people with lived experience. It has practical implications for future research as it highlights the importance of mixed-methods research in understanding lifestyle modification interventions and the need for population-specific improvements in the field (e.g., tailored interventions, development of evaluation instruments or tools, use of rigorous research methodologies and comprehensive reporting frameworks). Wide dissemination of related knowledge and the involvement of PPI groups, including people with lived experience, will help future researchers design interventions that consider the unique needs, desires and abilities of people with ID.

The effectiveness of prenatal and postnatal home visits by paramedical professionals and women's group meetings in improving maternal and child health outcomes in low and middle-income countries: a systematic review and meta-analysis.

OBJECTIVE: To assess the effectiveness of prenatal and postnatal home visits (HVs) and women group meetings (WGMs) by paramedical professionals to improve maternal and child health outcomes in low- and middle-income countries (LMICs). STUDY DESIGN: Systematic review and meta-analysis. METHODS: We conducted a systematic review of trials published till December 2020, as per registered protocol in The International Prospective Register of Systematic Reviews (PROSPERO) (CRD42018091968). Outcomes were neonatal mortality rate (NMR), maternal mortality ratio (MMR), the incidence of low birth weight, and still birth rate (SBR). The Cochrane Pregnancy and Childbirth Group's Trials Register, Cochrane Central Register of Controlled Trials, PubMed, and Excerpta Medica Database (EMBASE) were searched. Pooled results were estimated using random-effects meta-analysis in RevMan version 5.2. RESULTS: Twenty-five trials met the inclusion criteria. HVs were the key intervention in 12, WGMs in 11, and both interventions in 2 trials. The pooled estimates have shown that NMR was significantly reduced by HVs (OR 0.77, confidence interval [CI]: 0.67-0.90, P = 0.0007, I2 = 77%) and WGMs (OR 0.76, CI: 0.65-0.90, P = 0.001, I2 = 71%). SBR was significantly reduced by HVs (OR 0.77, CI: 0.70-0.85; P < 0.001, I2 = 0%). Subgroup analysis of studies in which more than 10% of pregnant women participated in the WGMs showed significant reduction in NMR (OR 0.67, CI 0.58-0.77, P = 0.00001, I2 = 31%) and MMR (OR 0.55, CI 0.36-0.84, P = 0.005, I2 = 27%). Two studies reported improvement in birth weight by HVs. CONCLUSIONS: HVs and WGMs (with >10% pregnant women) by paramedical professionals are effective strategies in reducing the NMR and MMR in LMICs. HVs were also effective in reducing SBR.

N-acetycysteine: A potential therapeutic agent in COVID-19 infection.

COVID-19 is an overwhelming pandemic which has shattered the whole world. Lung injury being the main clinical manifestation, it is likely to cause COPD (chronic obstructive pulmonary disease) and ARDS (acute respiratory distress syndrome). The possible cause behind this might be redox imbalance due to viral infection. Elevation in Glutathione (GSH) levels by administration of its promolecule might be effective. N-acetylcysteine is one such drug with potency to scavenge Reactive Oxygen Species, least side effects, and an effective precursor of glutathione. Consequently we hypothesize that N-acetylcysteine along with the conventional treatment may be treated as a potential therapeutic solution in cases of COVID-19 patients.

The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis.

OBJECTIVE: Skeletal muscle insulin signaling is a major determinant of muscle growth and glucose homeostasis. Protein kinase B/Akt plays a prominent role in mediating many of the metabolic effects of insulin. Mice and humans harboring systemic loss-of-function mutations in Akt2, the most abundant Akt isoform in metabolic tissues, are glucose intolerant and insulin resistant. Since the skeletal muscle accounts for a significant amount of postprandial glucose disposal, a popular hypothesis in the diabetes field suggests that a reduction in Akt, specifically in skeletal muscle, leads to systemic glucose intolerance and insulin resistance. Despite this common belief, the specific role of skeletal muscle Akt in muscle growth and insulin sensitivity remains undefined. METHODS: We generated multiple mouse models of skeletal muscle Akt deficiency to evaluate the role of muscle Akt signaling in vivo. The effects of these genetic perturbations on muscle mass, glucose homeostasis and insulin sensitivity were assessed using both in vivo and ex vivo assays. RESULTS: Surprisingly, mice lacking Akt2 alone in skeletal muscle displayed normal skeletal muscle insulin signaling, glucose tolerance, and insulin sensitivity despite a dramatic reduction in phosphorylated Akt. In contrast, deletion of both Akt isoforms (M-AktDKO) prevented downstream signaling and resulted in muscle atrophy. Despite the absence of Akt signaling, in vivo and ex vivo insulin-stimulated glucose uptake were normal in M-AktDKO mice. Similar effects on insulin sensitivity were observed in mice with prolonged deletion (4 weeks) of both skeletal muscle Akt isoforms selectively in adulthood. Conversely, short term deletion (2 weeks) of skeletal muscle specific Akt in adult muscles impaired insulin tolerance paralleling the effect observed by acute pharmacological inhibition of Akt in vitro. Mechanistically, chronic ablation of Akt induced mitochondrial dysfunction and activation of AMPK, which was required for insulin-stimulated glucose uptake in the absence of Akt. CONCLUSIONS: Together, these data indicate that chronic reduction in Akt activity alone in skeletal muscle is not sufficient to induce insulin resistance or prevent glucose uptake in all conditions. Therefore, since insulin-stimulated glucose disposal in skeletal muscle is markedly impaired in insulin-resistant states, we hypothesize that alterations in signaling molecules in addition to skeletal muscle Akt are necessary to perturb glucose tolerance and insulin sensitivity in vivo.

High fructose-induced metabolic changes enhance inflammation in human dendritic cells.

Dendritic cells (DCs) are critical antigen-presenting cells which are the initiators and regulators of the immune response. Numerous studies support the idea that dietary sugars influence DC functions. Increased consumption of fructose has been thought to be the leading cause of metabolic disorders. Although evidence supports their association with immune dysfunction, the specific mechanisms are not well understood. Fructose is one of the main dietary sugars in our diet. Therefore, here we compared the effect of fructose and glucose on the functions of human DCs. High levels of D-fructose compared to D-glucose led to activation of DCs in vitro by promoting interleukin (IL)-6 and IL-1ß production. Moreover, fructose exposed DCs also induced interferon (IFN)-γ secretion from T cells. Proinflammatory response of DCs in high fructose environment was found to be independent of the major known metabolic regulators or glycolytic control. Instead, DC activation on acute exposure to fructose was via activation of receptor for advanced glycation end product (RAGE) in response to increased accumulation of advanced glycation end products (AGE). However, chronic exposure of DCs to high fructose environment induced a shift towards glycolysis compared to glucose cultured DCs. Further investigations revealed that the AGEs formed by fructose induced increased levels of inflammatory cytokines in DCs compared to AGEs from glucose. In summary, understanding the link between metabolic changes and fructose-induced DC activation compared to glucose has broad implications for immune dysfunction associated with metabolic disorders.

Fructose-induced ROS generation impairs glucose utilization in L6 skeletal muscle cells.

High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing toward chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, glucose transporter type 4 (GLUT4) translocation, and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NF-κB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in the presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.

Isolated pancreatic transection in a case of blunt abdominal trauma.

A 20-year-old male patient presented with complaint of epigastric pain and vomiting, 1 day after an episode of bike handle injury. Ultrasound showed only mild free fluid in the abdomen. The patient gradually developed tachycardia, hypotension and guarding in the abdomen. CT scan revealed complete pancreatic transection between the neck and the body with segmental separation about an inch. Serum lipase was raised. Exploratory laparotomy revealed acute pseudocyst formation and necrotic slough on the transected ends. Distal pancreatectomy without splenectomy was performed. The patient recovered uneventfully.

Self retentive partial silicone auricular prosthesis: a case report.

An auricular prosthesis may be required for a number of conditions including congenital abnormalities, malignancy and trauma, which result in disfigurement of the pinna. Whatever the cause of the absence of the pinna, it is a significant loss of a prominent part of the face for the person involved. This article describes a simple and cost effective technique for retention of a silicone partial auricular prosthesis. A Fish-bone shaped substructure (FSS) designed and fabricated using orthodontic wire and autopolymerizing acrylic resin, was embedded into the silicone elastomer of a self-retentive silicone prosthesis. The prosthesis is designed to overcome the disadvantages associated with traditionally fabricated prostheses; namely poor structural strength, inadequate retention, poor adaptation and durability over time.

Glucose uptake stimulatory effect of 4-hydroxypipecolic acid by increased GLUT 4 translocation in skeletal muscle cells.

Peganum harmala Linn, commonly known as 'harmal' belonging to the family Zygophyllaceae, is one of the most important medicinal plants of India. In continuation of our drug development program on Indian medicinal plants we discovered antihyperglycemic activity in 4-hydroxypipecolic acid (4-HPA), isolated from the seed of P. harmala. Effect of 4-HPA on glucose uptake and glucose transporter-4 (GLUT-4) translocation was investigated in L6 skeletal muscle cell lines. Treatment with 4-HPA stimulated both glucose uptake and GLUT4 translocation from intracellular to cell surface in skeletal muscle cells in a concentration-dependent manner, which might be leading to antihyperglycemic effect.

The morphology and genetic characterization of Iheringascaris goai n. sp. (Nematoda: Raphidascarididae) from the intestine of the silver whiting and spotted catfish off the central west coast of India.

In this study a new species of nematode, Iheringascaris goai n. sp., is reported from two fish hosts, including silver whiting, Sillago sihama, and spotted catfish, Arius maculatus, caught off the Central West Coast of India at Goa. The new species can be differentiated morphologically from I. inquies, the most closely related species collected from cohabiting marine fish. The distinguishing characteristics are distinct cuticular striations, a unilateral excretory system, the presence of dentigerous ridges on the inner margin of the lips and the ratio of oesophagus to body length. In males, the ratio of spicules to body length is higher and the number of pre-anal papillae is less in comparison to those in I. inquies. In addition, the tail curves ventrad in males, while in females, the vulva is post-equatorial. The sequence alignment of 18S rDNA and cytochrome c oxidase subunit I with sequences of known species selected from the same superfamily shows a significant difference. The morphological and molecular differences reported here can, therefore, be used to assign the specimen to a new species.

Encoding of visual-spatial information in working memory requires more cerebral efforts than retrieval: Evidence from an EEG and virtual reality study.

Visual-spatial working memory tasks can be decomposed into encoding and retrieval phases. It was hypothesized that encoding of visual-spatial information is cognitively more challenging than retrieval. This was tested by combining electroencephalography with a virtual reality paradigm to observe the modulation in EEG activity. EEG power analysis results demonstrated an increase in theta activity during encoding in comparison to retrieval, whereas alpha activity was significantly higher for retrieval in comparison to encoding. We found that encoding required more cerebral efforts than retrieval. Further, as seen in fMRI studies, we observed an encoding/retrieval flip in that encoding and retrieval differentially activated similar neural substrates. Results obtained from sLORETA identified cortical sources in the inferior frontal gyrus, which is a part of dorsolateral prefrontal cortex (DLPFC) during encoding, whereas the inferior parietal lobe and precuneus cortical sources were identified during retrieval. We further tie our results into studies examining the default network, which have shown increased activation in DLPFC occurs in response to increased cerebral challenge, while posterior parietal areas show activation during baseline or internal processing tasks. We conclude that encoding of visual-spatial information via VR navigation task is more cerebrally challenging than retrieval.

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Human mesenchymal stem cells promote human osteoclast differentiation from CD34+ bone marrow hematopoietic progenitors.

Interactions between osteoclast progenitors and stromal cells derived from mesenchymal stem cells (MSCs) within the bone marrow are important for osteoclast differentiation. In vitro models of osteoclastogenesis are well established in animal species; however, such assays do not necessarily reflect human osteoclastogenesis. We sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human marrow-derived MSCs (hMSCs) and CD34+ hematopoietic stem cells (HSCs). After 3 weeks, coculture of hMSCs and HSCs resulted in an increase in hematopoietic cell number with formation of multinucleated osteoclast-like cells (Ocls). Coculture of hMSCs with HSCs, transduced with a retroviral vector that expresses enhanced green fluorescent protein, produced enhanced green fluorescent protein+ Ocls, further demonstrating that Ocls arise from HSCs. These Ocls express calcitonin and vitronectin receptors and tartrate-resistant acid phosphatase and possess the ability to resorb bone. Ocl formation in this assay is cell contact dependent and is independent of added exogenous factors. Conditioned medium from the coculture contained high levels of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), and macrophage-colony stimulating factor. IL-6 and LIF were present at low levels in cultures of hMSCs but undetectable in cultures of HSCs alone. These data suggest that coculture with HSCs induce hMSCs to secrete cytokines involved in Ocl formation. Addition of neutralizing anti-IL-6, IL-11, LIF, or macrophage-colony stimulating factor antibodies to the coculture inhibited Ocl formation. hMSCs seem to support Ocl formation as undifferentiated progenitor cells, because treatment of hMSCs with dexamethasone, ascorbic acid, and beta-glycerophosphate (to induce osteogenic differentiation) actually inhibited osteoclastogenesis in this coculture model. In conclusion, we have developed a simple and reproducible assay using culture-expanded hMSCs and purified HSCs with which to study the mechanisms of human osteoclastogenesis.

Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, tendon, and other connective tissues. These cells, referred to as mesenchymal stem cells, can be purified and culture-expanded from animals and humans and have been shown to regenerate functional tissue when delivered to the site of musculoskeletal defects in experimental animals. To test the ability of purified human mesenchymal stem cells to heal a clinically significant bone defect, mesenchymal stem cells isolated from normal human bone marrow were culture-expanded, loaded onto a ceramic carrier, and implanted into critical-sized segmental defects in the femurs of adult athymic rats. For comparison, cell-free ceramics were implanted in the contralateral limb. The animals were euthanized at 4, 8, or 12 weeks, and healing bone defects were compared by high-resolution radiography, immunohistochemistry, quantitative histomorphometry, and biomechanical testing. In mesenchymal stem cell-loaded samples, radiographic and histologic evidence of new bone was apparent by 8 weeks and histomorphometry demonstrated increasing bone formation through 12 weeks. Biomechanical evaluation confirmed that femurs implanted with mesenchymal stem cell-loaded ceramics were significantly stronger than those that received cell-free ceramics. These studies demonstrate that human mesenchymal stem cells can regenerate bone in a clinically significant osseous defect and may therefore provide an alternative to autogenous bone grafts.

Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation.

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB-10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB-10 antigen and its identification as activated leukocyte-cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 +/- 193 Da and that it possesses 17,763 +/- 237 Da of N-linked oligosaccharide substituents. Molecular cloning of a full-length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB-10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Mesenchymal stem cells in osteobiology and applied bone regeneration.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in dogs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.

Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.