Perspectives on oral chronic graft-versus-host disease from immunobiology to morbid diagnoses.

Chronic Graft-versus-Host Disease (cGVHD) is a major long-term complication, associated with morbidity and mortality in patients following allogenic hematopoietic cell transplantation (HCT) for immune hematopoietic disorders. The mouth is one of the most frequently affected organs after HCT (45-83%) and oral cGVHD, which may appear as the first visible sign. Manifestations present with mucosal lichenoid lesions, salivary gland dysfunction and limited oral aperture. Diagnosis of oral cGVHD severity is based on mucosal lesions with symptoms of sensitivity and pain and reduced oral intake. However, diagnostic difficulties arise due to subjective definitions and low specificity to cover the spectrum of oral cGVHD. In recent years there have been significant improvements in our understanding of the underlying oral cGVHD disease mechanisms. Drawing upon the current knowledge on the pathophysiology and biological phases of oral cGVHD, we address oral mucosa lichenoid and Sjogren's Syndrome-like sicca syndromes. We consider the response of alloreactive T-cells and macrophages to recipient tissues to drive the pathophysiological reactions and biological phases of acute inflammation (phase 1), chronic inflammation and dysregulated immunity (phase 2), and subsequent aberrant fibrotic healing (phase 3), which in time may be associated with an increased malignant transformation rate. When formulating treatment strategies, the pathophysiological spectrum of cGVHD is patient dependent and not every patient may progress chronologically through the biological stages. As such there remains a need to address and clarify personalized diagnostics and management to improve treatment descriptions. Within this review, we highlight the current state of the art knowledge on oral cGVHD pathophysiology and biological phases. We address knowledge gaps of oral cGVHD, with a view to facilitate clinical management and improve research quality on lichenoid biology and morbid forms of oral cGVHD.

A graph neural network framework for mapping histological topology in oral mucosal tissue.

BACKGROUND: Histological feature representation is advantageous for computer aided diagnosis (CAD) and disease classification when using predictive techniques based on machine learning. Explicit feature representations in computer tissue models can assist explainability of machine learning predictions. Different approaches to feature representation within digital tissue images have been proposed. Cell-graphs have been demonstrated to provide precise and general constructs that can model both low- and high-level features. The basement membrane is high-level tissue architecture, and interactions across the basement membrane are involved in multiple disease processes. Thus, the basement membrane is an important histological feature to study from a cell-graph and machine learning perspective. RESULTS: We present a two stage machine learning pipeline for generating a cell-graph from a digital H &E stained tissue image. Using a combination of convolutional neural networks for visual analysis and graph neural networks exploiting node and edge labels for topological analysis, the pipeline is shown to predict both low- and high-level histological features in oral mucosal tissue with good accuracy. CONCLUSIONS: Convolutional and graph neural networks are complementary technologies for learning, representing and predicting local and global histological features employing node and edge labels. Their combination is potentially widely applicable in histopathology image analysis and can enhance explainability in CAD tools for disease prediction.

Histopathological Grading of Oral Mucosal Chronic Graft-versus-Host Disease: Large Cohort Analysis.

Graft-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 eosin-stained 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.

Mesenchymal stromal cells modulate tissue repair responses within the injured vocal fold.

OBJECTIVES/HYPOTHESIS: This study aimed to determine whether local injection of human mesenchymal stromal cells (MSC) could modulate the early inflammatory response within injured vocal folds (VFs) to promote wound-healing processes. STUDY DESIGN: Experimental xenograft model. METHODS: VF injury was surgically induced by bilateral resection of the lamina propria of rabbits, and MSC were immediately injected into the injured area of both VFs. Animals were sacrificed on days 2, 4, and 24. Histological analyses were performed by hematoxylin and eosin, Masson's Trichrome, and elastin staining. Cell death was visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the M2 macrophage marker, CD163, detected by immunohistochemistry. Persistence of injected MSC was evaluated by fluorescent in situ hybridization (FISH). Quantitative polymerase chain reaction was performed on the contralateral VF. RESULTS: Histological examination at days 2 and 4 indicated that MSC were able to reduce tissue inflammation, with gene expression analysis confirming a significant reduction of proinflammatory markers, interleukin (IL)-1ß, and IL-8. FISH demonstrated low-level persistence of injected MSC at both time points, and TUNEL confirmed localized cell death at the injury site. Increased levels of CD163+ anti-inflammatory macrophages indicated a change in the immune milieu, supporting wound resolution. Evidence of a more organized collagen matrix suggests that MSC may enhance the production of a functional repair tissue after injury, despite their low-level persistence within the tissue. CONCLUSIONS: This study demonstrates that MSC are able to positively modulate the early wound-healing response through resolution of the inflammatory phase and promotion of tissue repair. LEVEL OF EVIDENCE: NA Laryngoscope, 130:E21-E29, 2020.

Tissue immune profiles supporting response to mesenchymal stromal cell therapy in acute graft-versus-host disease-a gut feeling.

Acute graft-versus-host disease (aGvHD), post-allogeneic hematopoietic stem cell transplantation, is associated with high mortality rates in patients not responding to standard line care with steroids. Adoptive mesenchymal stromal cell (MSC) therapy has been established in some countries as a second-line treatment.Limitations in our understanding as to MSC mode of action and what segregates patient responders from non-responders to MSC therapy remain. The principal aim of this study was to evaluate the immune cell profile in gut biopsies of patients diagnosed with aGvHD and establish differences in baseline cellular composition between responders and non-responders to subsequent MSC therapy.Our findings indicate that a pro-inflammatory immune profile within the gut at the point of MSC treatment may impede their therapeutic potential. These findings support the need for further validation in a larger cohort of patients and the development of improved biomarkers in predicting responsiveness to MSC therapy.

Dynamic Changes in Brain Mesenchymal Perivascular Cells Associate with Multiple Sclerosis Disease Duration, Active Inflammation, and Demyelination.

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 (PDGFRß), CD73, CD271, alpha-smooth 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+ PDGFRß+ Ki67+ cells and CD73+ CD271+ PDGFRß+ 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+ PDGFRß+ Ki67+ and CD73+ CD271+ PDGFRß+ Ki67- MPC subpopulations compared to inactive lesions. Chronic lesions presented with decreased levels of CD146+ PDGFRß+ Ki67+ MPC cells compared to control tissue. Furthermore, LPMS lesions displayed increased numbers of blood vessels harboring greatly enlarged CD73+ CD271+ 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.

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

OBJECTIVE: Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. MATERIALS AND METHODS: Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E2, interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1ß stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. RESULTS: PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p < .001). Short-term exposure to 0.005% PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p < .001). IL-1ß stimulation induced an inflammatory response with a significant increase in the secretion of PGE2, IL-6, IL-8 and MMP-1. Treatment of IL-1ß stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE2, IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE2, IL-6, IL-8 or MMP-1 by gingival fibroblasts. CONCLUSIONS: Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1ß-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

The glycosylation profile of osteoadherin alters during endochondral bone formation.

Endochondral bone formation involves the dynamic interplay between the cells and their extracellular environment to facilitate the deposition of a calcified matrix. Numerous molecules are involved within this process, including collagens and non-collagenous proteins, and their post-translational modifications have been shown to effect their biomolecular interactions. Osteoadherin (OSAD), a keratin sulfate (KS)-substituted small leucine-rich proteoglycan has been isolated from mineralized tissues and is considered to be a mineralized tissue-specific protein. However, to date, information is limited concerning the dynamic expression and role of this proteoglycan during bone formation and the biomineralization process. The current study aimed to examine the dynamic expression of this protein throughout mouse metatarsal long bone development, from the cartilage anlagen (E15) to the fully formed bone (Adult). Using quantitative gene expression analysis we observed that OSAD was produced with the onset of mineralization and the formation of the ossification center. This finding was reflected in the localization studies, using both light and electron microscopy, and showed that initial OSAD localization was restricted to the endosteal surfaces of the diaphysis and forming metaphysis. Furthermore, we analyzed protein extracts, both mineral and non-mineral associated fractions, and showed that OSAD was substituted with varying patterns of glycosylation during bone development. Sequential enzymatic digestions of the non-mineral bound protein extracts demonstrated that OSAD lacked any KS chains throughout all development stages. Whereas, in the mineral bound fractions, with long bone maturation the substitution with KS became more apparent with development. Therefore, it can be concluded that different pools of OSAD are produced during endochondral bone formation and these may have specific roles in directing the mineralization process.

Osteoadherin accumulates in the predentin towards the mineralization front in the developing tooth.

BACKGROUND: Proteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization. CONCLUSIONS: These studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.

Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, beta-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.

Osteoadherin is upregulated by mature osteoblasts and enhances their in vitro differentiation and mineralization.

During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.

Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.

Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

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.

Substitution of bovine dentine sialoprotein with chondroitin sulfate glycosaminoglycan chains.

Dentine sialoprotein (DSP) represents 5-8% of all non-collagenous proteins present in the tooth, but, together with dentine phosphoprotein, has been shown to be vital for correct tooth formation. Recently, the existence of a highly glycosylated form of porcine DSP has been reported and it was shown to possess glycosaminoglycan (GAG) chains. The current investigation confirms that this is also the case for bovine DSP and has further characterized these carbohydrates. Dentine sialoprotein was purified from bovine dentine extracts by anion exchange chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectroscopy. An increase in molecular mass was observed, from 120 kDa to greater than 250 kDa, with a corresponding rise in anionic strength. Cellulose acetate electrophoresis and western blotting indicated the presence of chondroitin sulfate GAG chains within these dentine fractions. Further examination using sequential digestion with chondroitinase AC and N-glycosidase cleaved the samples first to 95 kDa and then to 80 kDa, respectively, confirming a high level of glycosylation. These results support the classification of bovine DSP as a proteoglycan, and that the carbohydrate substitutions may contribute to the functional properties of DSP.

Modulation of collagen fibrillogenesis by dentinal proteoglycans.

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.

Nucleobindin is produced by bone cells and secreted into the osteoid, with a potential role as a modulator of matrix maturation.

Nucleobindin (Nuc), also known as CALNUC, is a Ca(2+)-binding protein, located in the nucleus, the Golgi apparatus and the endoplasmic reticulum (ER). The presence of a signal sequence in Nuc suggests secretion from the cell and it has been found in bone extracellular matrix. Within the present study, molecular biological and morphological methods were combined to evaluate the synthesis and distribution of Nuc in and around cells of rat metaphyseal and calvarial bone. Northern blot analysis and in situ hybridization of bone tissues confirmed that the protein was a product of bone cells. By electron microscopy, immunolabeling for Nuc was seen in osteoid of newly formed bone, on all surfaces facing the various bone cells and also in compact bone. Intracellularly, the gold particles were found in the rough ER of osteoblasts, which suggested synthesis of the protein by these cells. Compared to bone sialoprotein and osteopontin, Nuc demonstrated different localization pattern in bone trabeculae, with the majority of labeling restricted to nonmineralized osteoid. Moreover, the role of Nuc during the mineralization process was investigated in rat calvaria-derived primary osteoblasts grown under osteogenic conditions. Semiquantitative RT-PCR and Northern blot analysis showed Nuc expression to be low during cell proliferation, upregulated during differentiation and matrix maturation, but subsequently downregulated during mineralization. In summary, our data show that Nuc was synthesized by osteoblasts and osteocytes, and secreted into the osteoid, suggesting a role as a modulator of matrix maturation in the mineralization process in bone.

The interaction of recombinant decorin with alpha2HS-glycoprotein-implications for structural and functional investigations.

Isolated protein preparations of the small leucine-rich proteoglycans (SLRPs) associated with mineralized tissues have provided important information in understanding their structural and functional interactions within extracellular matrices and their potential roles in mineralization. Two important SLRPs, decorin and biglycan, copurify following extraction and purification from mineralized tissues using standard procedures, and to overcome this problem decorin was synthesized within a mammalian expression system to obtain pure preparations. The expressed protein was purified from the culture medium using anion-exchange chromatography, and characterization confirmed the presence of a decorin-rich fraction. However, N-terminal sequencing revealed the additional presence of alpha2HS-glycoprotein (alpha2HSG), representing approximately 35% of the total purified fraction. The decorin-rich fraction was subjected to selected further purification techniques to separate decorin from alpha2HSG. Application of the sample at a low concentration (1 mg/ml) to a second anion-exchange procedure and elution over an expanded sodium chloride gradient resulted in a high degree of purity (98%), with a single protein isolate demonstrable by SDS-PAGE. Electroelution achieved partial purification ( approximately 89%), but immunoprecipitation with antibodies against the glycosaminoglycan chain and the polyhistidine tag failed to separate the two proteins. This study suggests there is a strong interaction between recombinantly produced decorin and alpha2 HSG and highlights the importance of the purification technique to the application of recombinantly produced proteins or those that have been extracted from mineralized tissues for use in structural and functional interactions.