This paper reviews our current state of knowledge of the roles the small leucine-rich proteoglycans (SLRPs) play in the formation of connective tissue and mineralised tissue matrices. Both, the SLRPs biglycan and decorin are highly expressed in extracellular bone matrix and there is now substantial evidence to support an increasing role for biglycan and decorin in influencing bone cell differentiation and proliferative activity. In addition decorin and biglycan have been implicated in regulating mineral deposition and crystal morphology, whilst decorin has also roles in organic matrix assembly. In order to further assess the role of these SLRPs during bone formation we have initiated studies investigating primary bone cell culture models from rats (bone marrow stromal cells, and bone cells from alveolar bone explants), and identified periods relating to cell proliferation, organic matrix deposition, remodelling of the osteoid, and mineral deposition. Analysis of mRNA levels and the nature of the proteoglycan demonstrated that dermatan sulphate substituted biglycan was expressed during phases relating to cell proliferation, ceased at early matrix deposition, and then biglycan was re-expressed at the onset of mineralisation, but was conjugated to chondroitin sulphate. Decorin was expressed later than biglycan, was associated with early matrix deposition, but then continued to the mineralisation stages. Again, dermatan sulphate-decorin prevailed earlier within osteoid matrix, whilst chondroitin sulphate-decorin predominated later within the mineralizing matrix. The nature of the GAG chain conjugated to SLRP and the timing of its expression would seem to dictate the functions biglycan and decorin play in bone formation.

Information concerning the development and differentiation of human embryonic stem (hES) cells in vivo is limited. The present study has focused on the in vivo outcome and differentiation of the hESC line HS181, after injection into SCID/beige mice. hES cell-derived teratomas were explored using histological evaluation and by the identification of markers for differentiated cells and tissues. The analyses identified predominant differentiation along a neuronal lineage, the formation of bone/cartilage and epithelia. Fluorescent in situ hybridization (FISH) analysis with a human-specific probe showed the teratomas to be mainly of human origin, with the most organized areas being exclusively human. Importantly, the study revealed interactions between mouse and human tissues, most notably in the formation of vessels. Both mouse and human cells contributed to specific microstructures in which mouse cells could be observed to take on the appropriate histiotypic appearance. Hence, HS181 cells were able to develop into defined mature tissues, supporting the relevant use of this hES cells model for studies of early human development, given the use of appropriate controls for host contribution. Although extensive mitotic activity implicated progenitor cell activity, no detectable multipotent or malignant areas were observed during the observation period. Persisting undifferentiated hESC were not detected.

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TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

versus-host disease (GVHD) can manifest as acute or chronic complications in patients after hematopoietic cell transplantation (HCT). Oral chronic GVHD (cGVHD) occurs in approximately 70% of HCT recipients and includes lichenoid-like mucosal reactions, restricted mouth opening, and salivary gland dysfunction. However, the underlying histopathological presentation remains to be validated in large cohorts. We characterized the histopathological features of oral mucosal cGVHD and devised a scoring model in a large patient cohort (n = 112). Oral mucosal biopsy sections (n = 303) with and without oral cGVHD were identified from archived and current HCT recipients with additional healthy controls. Histological screening was performed on hematoxylin and eosinstained and periodic acid-Schiff-stained sections. A points-based grading tool (0 to 19, grade 0 to IV) was established based on intraepithelial lymphocytes and band-like inflammatory infiltrate, atrophic epithelium with basal cell liquefaction degeneration, including apoptosis, as well as separation of epithelium and pseudo-rete ridges. Validation involved 62 biopsy specimens, including post-HCT (n = 47) and healthy (n = 15) specimens. Remaining biopsy specimens (n = 199) were blindly graded by 3 observers. Histological severity was correlated with clinical diagnostic and distinctive features, demonstrating a spectrum of individual patient severity, including frequent signs of subclinical GVHD in healthy mucosa. However, oral cGVHD presented with significantly higher (P < .001) scores compared with HCT controls, with moderate to high positive likelihood ratios for inflammatory infiltrate, exocytosis, and basal membrane alterations. The grade II-IV biopsy specimens demonstrated a histopathological diagnosis of active mucosal lichenoid-like cGVHD, highlighting the importance of correlating clinical presentation with the dynamic histopathological processes for improved patient stratification. In addition, this tool could be used for assessing treatments, pathological processes, and immune cellular content to provide further insight into this debilitating disease.

Studies have identified different pools of proteoglycan (PG) species present within the unmineralized matrix of the predentine, the transitional phase at the predentine-dentine interface and the mineralized dentine. These PGs alter with respect to the chemical nature of their glycosaminoglycan (GAG) chains and as a result of extracellular processing of the macromolecule in the matrix. The present study has examined the influence of the PGs isolated from these phases and the influence of the attached GAG chains, upon their ability to control collagen fibrillogenesis. PGs isolated from the three phases were characterized and determined to contain a mixture of decorin and biglycan. Results have indicated that predentine PGs, which are substituted with a higher proportion of dermatan sulfate, significantly delayed fibril formation while ultimately promoting the formation of thicker fibrils. Removal of the GAG chains further delayed fibrillogenesis, leading to the formation of thinner fibrils, compared with the collagen-only control. PGs isolated from predentine-dentine, which contained a higher proportion of chondroitin sulfate, also significantly delayed fibrillogenesis, resulting in thicker collagen fibrils. GAG chains attached to the predentine-dentine interface PGs played a role in the timing of fibrillogenesis with fibril formation initiated at the same time as the collagen control, but yielding thicker fibrils. Dentine PGs significantly inhibited fibrillogenesis and fibril thickness over concentrations of 50-25 microg/mL protein. In conclusion, the PGs isolated from the distinct phases have indicated differing roles in the orchestrated organization of the extracellular matrix during dentinogenesis, with roles for both the core protein and attached GAG chains indicated.

Vascular changes, including blood brain barrier destabilization, are common pathological features in multiple sclerosis (MS) lesions. Blood vessels within adult organs are reported to harbor mesenchymal stromal cells (MSCs) with phenotypical and functional characteristics similar to pericytes. We performed an immunohistochemical study of MSCs/pericytes in brain tissue from MS and healthy persons. Post-mortem brain tissue from patients with early progressive MS (EPMS), late stage progressive MS (LPMS), and healthy persons were analyzed for the MSC and pericyte markers CD146, platelet-derived growth factor receptor beta (PDGFRb), CD73, CD271, alphasmooth muscle actin, and Ki67. The MS samples included active, chronic active, chronic inactive lesions, and normal-appearing white matter. MSC and pericyte marker localization were detected in association with blood vessels, including subendothelial CD146 1 PDGFRb 1 Ki67 1 cells and CD73 1 CD271 1 PDGFRb 1 Ki67 -cells within the adventitia and perivascular areas. Both immunostained cell subpopulations were termed mesenchymal perivascular cells (MPCs). Quantitative analyses of immunostainings showed active lesions containing increased regions of CD146 1 PDGFRb 1 Ki67 1 and CD73 1 CD271 1 PDGFRb 1 Ki67 -MPC subpopulations compared to inactive lesions. Chronic lesions presented with decreased levels of CD146 1 PDGFRb 1 Ki67 1 MPC cells compared to control tissue. Furthermore, LPMS lesions displayed increased numbers of blood vessels harboring greatly enlarged CD73 1 CD271 1 adventitial and perivascular areas compared to control and EPMS tissue. In conclusion, we demonstrate the presence of MPC subgroups in control human brain vasculature, and their phenotypic changes in MS brain, which correlated with inflammation, demyelination and MS disease duration. Our findings demonstrate that brain-derived MPCs respond to pathologic mechanisms involved in MS disease progression and suggest that vessel-targeted therapeutics may benefit patients with progressive MS. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1840-1851 SIGNIFICANCE STATEMENTThis immunohistochemical study characterized vascular cells phenotypically similar to pericytes and mesenchymal stromal cells (MSCs) in brain tissue of multiple sclerosis (MS) and healthy persons. We found variations in phenotype, distribution, and proliferation of cells co-expressing markers for MSCs and pericytes that associated with inflammation and MS disease duration. Tissue from long standing MS presented with increased numbers of blood vessels that harbored expanded perivascular areas of cells staining positive for MSCs/pericyte markers. This is the first study to show involvement of MSC/pericyte phenotypic changes in MS pathology and highlights that vessel-targeted treatments may benefit late stage MS.

Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.