Fibular head avulsion fractures accompanying operative treated medial tibial plateau fractures.

OBJECTIVE: The aims of this work are to determine how frequently medial tibial plateau fractures are accompanied by fibular head avulsion fractures and evaluate the sensitivity of radiographs detecting them, and also to assess if the presence of fibular fracture is correlated with long-term functional outcome and peroneal nerve damage. MATERIALS AND METHODS: A retrospective chart review of operated patients with medial tibial plateau fractures at level I trauma center during 2002-2008 was performed. From 63 patients imaged preoperatively, 59 had CT and radiographs, three had only CT, and one only radiograph. The presence and fragment size of fibular fracture were retrospectively evaluated. Body mass index (BMI) and functional outcome measurements (the Modified Lysholm knee score and WOMAC) were available for 46 patients. RESULTS: Fourteen out of 63 patients (22.2%) had fibular fractures. Of the 59 patients with both CT and radiographs, 12 had fibular fractures, and of these, nine were seen with both modalities and three only in CT. Functional scores were available for ten patients with fibular fracture. Patients with fibular fracture seen on radiographs had a significantly higher score on WOMAC function (26 vs. 7; p = 0.027). The patients with fibular fractures had also higher BMI (p = 0.035). Of the six patients with peroneal nerve damage, 50% had fibular fracture. CONCLUSIONS: In patients with operatively treated medial tibial plateau fracture, the fibular fractures are relatively common. Detecting it is important, as it may be associated with worse functional scores and peroneal nerve paresis. Some fibular fractures may remain undetected on radiographs, hence preoperative CT is recommended.

Extension Block Pinning for Unstable Proximal Interphalangeal Joint Dorsal Fracture Dislocations.

PURPOSE: To evaluate the outcomes of extension block pinning used to treat unstable dorsal fracture dislocations of the proximal interphalangeal (PIP) joint. The factors affecting the functional outcome were analyzed. METHODS: A series of 53 patients with 55 dorsal fracture dislocations of the PIP joint treated with closed reduction and extension block pinning were retrospectively reviewed. Additional percutaneous intramedullary fracture reduction (16 cases) or open fracture reduction (4 cases) had been performed. The radiological and clinical evaluations were included. RESULTS: At a mean follow-up of 5.2 years (range, 1.0-10.6 years), 39 patients with 41 injured fingers were evaluated. The fracture fragments involved 30% to 69% (mean, 50%) of the articular surface of the middle phalanx. The mean range of motion was 80° (range, 35° to 115°) at the PIP joint with a mean extension loss of 6° (range, 0° to 50°) excluding 2 joints that were salvaged with arthrodesis. The mean range of motion of the distal interphalangeal joint was 68° (range, 5° to 90°). The mean visual analog scale for digit pain was 1.5/10. The reduction of the joint was achieved intraoperatively in all cases. However, after the hardware removal, recurrent minimal subluxation was observed in 12 cases (29%). Recurrent subluxation was associated with increased residual pain. The length of follow-up time had a positive correlation, whereas the patient age had a negative correlation with the range of motion of the injured PIP joint. CONCLUSIONS: The extension block pinning technique is a simple and valuable technique for treating unstable dorsal PIP fracture-dislocation injuries producing satisfactory long-term results.

Histamine transport and metabolism are deranged in salivary glands in Sjogren's syndrome.

OBJECTIVE: To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS: Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS: mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION: Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to ∼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 µM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

BACKGROUND AND PURPOSE: Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. METHODS: We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. RESULTS: In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166(×) progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). INTERPRETATION: Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation.

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.

Adhesion, spreading and osteogenic differentiation of mesenchymal stem cells cultured on micropatterned amorphous diamond, titanium, tantalum and chromium coatings on silicon.

It was hypothesized that human mesenchymal stromal cell (hMSC) can be guided by patterned and plain amorphous diamond (AD), titanium (Ti), tantalum (Ta) and chromium (Cr) coatings, produced on silicon wafer using physical vapour deposition and photolithography. At 7.5 h hMSCs density was 3.0-3.5 x higher (P < 0.0003, except Ti) and cells were smaller (68 vs. 102 microm, P 0.000006-0.02) on patterns than on silicon background. HMSC-covered surface of the background silicon was lower on Ti than AD patterns (P = 0.015), but at 5 days this had reversed (P = 0.006). At 7.5 h focal vinculin adhesions and actin cytoskeleton were outgoing from pattern edges so cells assumed geometric square shapes. Patterns allowed induced osteogenesis, but less effectively than plain surfaces, except for AD, which could be used to avoid osseointegration. All these biomaterial patterns exert direct early, intermediate and late guidance on hMSCs and osteogenic differentiation, but indirect interactions exist with cells on silicon background.

BMPs in periprosthetic tissues around aseptically loosened total hip implants.

BACKGROUND AND PURPOSE: Primary and dynamically maintained periprosthetic bone formation is essential for osseointegration of hip implants to host bone. Bone morphogenetic proteins (BMPs) play a role in osteoinductive bone formation. We hypothesized that there is an increased local synthesis of BMPs in the synovial membrane-like interface around aseptically loosened total hip replacement (THR) implants, as body attempts to generate or maintain implant fixation. PATIENTS AND METHODS: We compared synovial membrane-like interface tissue from revised total hip replacements (rTHR, n = 9) to osteoarthritic control synovial membrane samples (OA, n = 11. Avidin-biotin-peroxidase complex staining and grading of BMP-2, BMP-4, BMP-6, and BMP-7 was done. Immunofluorescence staining was used to study BMP proteins produced by mesenchymal stromal/stem cells (MSCs) and osteoblasts. RESULTS AND INTERPRETATION: All BMPs studied were present in the synovial lining or lining-like layer, fibroblast-like stromal cells, interstitial macrophage-like cells, and endothelial cells. In OA and rTHR samples, BMP-6 positivity in cells, inducible by the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta, predominated over expression of other BMPs. Macrophage-like cells positive for BMP-4, inducible in macrophages by stimulation with particles, were more frequent around loosened implants than in control OA samples, but apparently not enough to prevent loosening. MSCs contained BMP-2, BMP-4, BMP-6, and BMP-7, but this staining diminished during osteogenesis, suggesting that BMPs are produced by progenitor cells in particular, probably for storage in the bone matrix.

Intracrine androgenic apparatus in human bone marrow stromal cells.

It was suggested that human mesenchymal stromal cells might contain an intracrine enzyme machinery potentially able to synthesize the cell's own supply of dihydrotestosterone (DHT) from dehydroepiandrosterone (DHEA) pro-hormone produced in the adrenal cortex in the reticular zone, which is unique to primates. Indeed, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 5alpha-reductase enzyme proteins were expressed in resting mesenchymal stromal cells (MSCs) in vitro. However, the 'bridging' enzymes 17beta-HSDs, catalysing interconversion between 17beta-ketosteroids and 17beta-hydroxysteroids, were not found in resting MSCs, but 17beta-HSD enzyme protein was induced in a dose-dependent manner by DHEA. Quantitative real-time polymerase chain reactions disclosed that this was mainly due to induction of the isoform 5 catalysing this reaction in 'forward', androgen-bound direction (P < 0.01). This work demonstrates that the MSCs have an intracrine machinery to convert DHEA to DHT if and when challenged by DHEA. DHEA as substrate exerts a positive, feed-forward up-regulation on the 17beta-hydroxy steroid dehydrogenase-5, which may imply that DHEA-DHT tailor-making in MSCs is subjected to chronobiological regulation.

Microbial antigens mediate HLA-B27 diseases via TLRs.

HLA-B27 positive individuals are predisposed to reactive arthritis developing 1-3 weeks after urogenital and gastrointestinal infections. Also ankylosing spondylitis (AS) associates strongly to HLA-B27, but no specific infection, Klebsiella pneumoniae excluded, has been linked to it. Before the discovery of its HLA-B27 association there were many reports suggesting a link between chronic prostatitis in men or pelvic inflammatory disease in women and AS. They have since been forgotten although HLA-B27 did not help to understand, why this disease has an axial and ascending nature. It is proposed that the urogenital organs form a source of damage (or danger)-associated molecular patterns (DAMPs), either exogenous pathogen-associated molecular patterns (PAMPs) from microbes or endogenous alarmins, such as uric acid, released from necrotic cells or urate deposits. DAMPs are slowly seeded from low-down upwards via the pelvic and spinal lymphatic pathways. They reach Toll-like receptors (TLRs) in their target mesenchymal stem cells, which are stimulated to ectopic enchondral bone formation leading to syndesmophytes and bamboo spine. At the same time inflammatory cytokines induce secondary osteoporosis of the spine. This new paradigm places microbes, HLA-B27 and TLRs in the pathogenic centre stage, but without pinpointing any (one) specific pathogen; instead, shared microbial patterns are indicated.

Human osteoblasts produce cathepsin K.

Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H(+)-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively. One of the main collagenolytic proteinase in osteoclasts is cathepsin K, a member of papain family cysteine proteinases. Recently, it has been shown that osteoblasts may contribute to organic matrix remodeling. We therefore investigated their ability to produce cathepsin K for this action. Trabecular bone samples were collected from patients operated due to a fracture of the femoral neck. Part of the bone was decalcified and the rest was used for cell isolation. Sections from the decalcified bone were immunostained with antibodies against cathepsin K. Isolated cells were characterized for their ability to form mineralized matrix and subsequently analyzed for their cathepsin K production by Western blotting and quantitative RT-PCR. Osteoblasts, bone lining cells and some osteocytes in situ showed cathepsin K immunoreactivity and osteoblast-like cells in vitro produced cathepsin K mRNA and released both 42 kDa pro- and 27 kDa processed cathepsin K to culture media. Osteoblastic cathepsin K may thus contribute to collagenous matrix maintenance and recycling of improperly processed collagen I. Whether osteoblastic cathepsin K synthesis has consequences in diseases characterized by abnormal bone matrix turnover remains to be investigated.

Screening for celiac disease in Down's syndrome patients revealed cases of subtotal villous atrophy without typical for celiac disease HLA-DQ and tissue transglutaminase antibodies.

AIM: To investigate the prevalence of celiac disease (CD) as well as CD marker antibodies and susceptibility HLA-DQ haplotypes in 134 karyotyped Down's syndrome (DS) patients. METHODS: Immunoglobulin A (IgA) and G (IgG) type anti-gliadin antibodies (AGA), IgA type anti-tissue transglutaminase (tTG) antibodies (anti-tTG) with antigen of guinea pig and human source were determined by enzyme-linked immunosorbent assay and endomysium antibodies (EMA) by indirect immunofluorescence test. HLA-DQA1*0501/DQB1*0201 (DQ2) was revealed by polymerase chain reaction. Celiac disease was diagnosed by revised ESPGHAN criteria. RESULTS: 41% of DS patients had AGA, 6.0% IgA anti-tTG with guinea pig antigen, and 3.0% IgA EMA (all positive for anti-tTG with human tTG). Subtotal villous atrophy was found in 5 out of 9 DS patients who had agreed to small bowel biopsy. One of them had DQA1*0501/DQB1*0201 and anti-tTG and EMA i.e. typical for CD markers (this case also fulfilled the ESPGHAN diagnostic criteria),but other four lacked these markers. Three non-biopsied DS patients had also most probably CD because DQA1*0501/DQB1*0201 and IgA anti-tTG (EMA) were detected. Thus, the prevalence of CD among our DS patients population is 3.0% (95 % of confidence interval [CI]: 0.1-5.9%). CONCLUSION: We confirm the increased frequency of CD among DS patients. In addition, we have revealed a subgroup of patients with subtotal villous atrophy but without characteristic for CD immunological and genetic markers. Whether these cases represent CD (with atypical immunopathogenesis) or some other immune enteropathy, requires further investigations.

A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair.

The aim was to develop a hybrid three-dimensional-tissue engineering construct for chondrogenesis. The hypothesis was that they support chondrogenesis. A biodegradable, highly porous polycaprolactone-grate was produced by solid freeform fabrication. The polycaprolactone support was coated with a chitosan/polyethylene oxide nanofibre sheet produced by electrospinning. Transforming growth factor-ß3-induced chondrogenesis was followed using the following markers: sex determining region Y/-box 9, runt-related transcription factor 2 and collagen II and X in quantitative real-time polymerase chain reaction, histology and immunostaining. A polycaprolactone-grate and an optimized chitosan/polyethylene oxide nanofibre sheet supported cellular aggregation, chondrogenesis and matrix formation. In tissue engineering constructs, the sheets were seeded first with mesenchymal stem cells and then piled up according to the lasagne principle. The advantages of such a construct are (1) the cells do not need to migrate to the tissue engineering construct and therefore pore size and interconnectivity problems are omitted and (2) the cell-tight nanofibre sheet and collagen-fibre network mimic a cell culture platform for mesenchymal stem cells/chondrocytes (preventing escape) and hinders in-growth of fibroblasts and fibrous scarring (preventing capture). This allows time for the slowly progressing, multiphase true cartilage regeneration.

A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.

Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

INTRODUCTION: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally. METHODS: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified. RESULTS: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP. CONCLUSIONS: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

Identification of histamine receptor subtypes in skeletal myogenesis.

To date, conventional and/or novel histamine receptors (HRs) have not been investigated in mouse skeletal myogenesis. Therefore, the present study aimed to investigate the HR­subtypes in skeletal myogenesis. The myogenesis of C2C12 skeletal myoblasts was evaluated using desmin, myogenin and myosin heavy chain (Myh) as early, intermediate and late differentiation markers, respectively. Reverse transcription­quantitative polymerase chain reaction and immunostaining were performed and the messenger RNA (mRNA) expression levels of the HR­subtypes and markers were determined. H1R mRNA was found to be highly expressed in myoblasts at day 0; however, the expression levels were reduced as differentiation progressed. By contrast, H2R mRNA expression remained constant, while H3R mRNA expression increased by 28­, 103­ and 198­fold at days 2, 4 and 6 compared with the baseline level (day 0), respectively. In addition, Myh expression increased by 7,718­, 94,487­ and 286,288­fold on days 2, 4 and 6 compared with the baseline expression level (day 0). Weak positive staining of the cells for H3R protein was observed on day 2, whereas highly positive staining was observed on days 4 and 6. HR expression during myogenesis was, in part, regulated by the stage of differentiation. These results along with previous findings indicated possible involvement of H1R in the regulation of progenitor cell mitogenesis and of H2R in the relaxation of acetylcholine­stimulated contraction of muscle cells, following the activation of professional histamine­producing cells, including mast cells. By contrast, H3R may participate in the regulation of specialized myocyte functions, potentially by maintaining the relaxed state under the influence of constitutive H3R activity and low histamine concentrations, locally produced/released by non­professional histamine­producing cells.

Sample processing, protocol, and statistical analysis of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of protein, cell, and tissue samples.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is increasingly being used in analysis of biological samples. For example, it has been applied to distinguish healthy and osteoarthritic human cartilage. This chapter discusses ToF-SIMS principle and instrumentation including the three modes of analysis in ToF-SIMS. ToF-SIMS sets certain requirements for the samples to be analyzed; for example, the samples have to be vacuum compatible. Accordingly, sample processing steps for different biological samples, i.e., proteins, cells, frozen and paraffin-embedded tissues and extracellular matrix for the ToF-SIMS are presented. Multivariate analysis of the ToF-SIMS data and the necessary data preprocessing steps (peak selection, data normalization, mean-centering, and scaling and transformation) are discussed in this chapter.

Label-free imaging of adipogenesis by coherent anti-stokes Raman scattering microscopy.

Label-free imaging technologies to monitor the events associated with early, intermediate and late adipogenic differentiation in multipotent mesenchymal stromal cells (MSCs) offer an attractive and convenient alternative to conventional fixative based lipid dyes such as Oil Red O and Sudan Red, fluorescent labels such as LipidTOX, and more indirect methods such as qRT-PCR analyses of specific adipocyte differentiation markers such as peroxisome PPARγ and LPL. Coherent anti-Stokes Raman scattering (CARS) microscopy of live cells is a sensitive and fast imaging method enabling evaluation of the adipogenic differentiation with chemical specificity. CARS microscopy is based on imaging structures of interest by displaying the characteristic intrinsic vibrational contrast of chemical bonds. The method is nontoxic, non-destructive, and minimally invasive, thus presenting a promising method for longitudinal analyses of live cells and tissues. CARS provides a coherently emitted signal that is much stronger than the spontaneous Raman scattering. The anti-Stokes signal is blue shifted from the incident wavelength, thus reducing the non-vibrational background present in most biological materials. In this chapter, we aim to provide a detailed approach on how to induce adipogenic differentiation in MSC cultures, and present our methods related to label-free CARS imaging of the events associated with the adipogenesis.

Osteogenic differentiation on DLC-PDMS-h surface.

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion-growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use.