ICAM-1-mediated osteoblast-T lymphocyte direct interaction increases mineralization through TGF-ß1 suppression.

Modulation of osteoblast functions by T lymphocytes is important in inflammation-associated mineralized tissue diseases. The study aimed to determine whether direct interaction between these two cell types affects osteoblast functions and mineralization. The results showed that direct contact between the two cell types was evident by scanning electron microscopy and transmission electron microscopy. Under osteogenic induction, higher hydroxyapatite precipitation was observed in cocultures with direct contact with T lymphocytes compared with that by osteoblasts cultured alone. Cocultures without direct cell contact caused a decrease in mineralization. Direct cell contact also upregulated intercellular adhesion molecule (ICAM)-1 and simultaneously downregulated transforming growth factor (TGF)-ß1 in osteoblasts. However, the downregulation of TGF-ß1 was reversed by ICAM-1 blocking. Exogenously added TGF-ß1 in cocultures with direct cell contact suppressed mineralization. In conclusion, studies are consistent with ICAM-1-mediated direct contact between osteoblasts and T lymphocytes increasing mineralization via downregulation of TGF-ß1 in osteoblasts in vitro. This suggests a possible unexpected, but crucial, role of T lymphocytes in enhancing matrix mineralization during the repair process in vivo. The study identifies ICAM-1/TGF-ß1 as possible novel therapeutic targets for the treatment and prevention of inflammation-associated mineralized tissue diseases.

Interaction of enamel matrix proteins with human periodontal ligament cells.

OBJECTIVES: It has recently been shown that enamel matrix derivative (EMD) components (Fraction C, containing <6 kDa peptides (mainly a 5.3 kDa tyrosine-rich amelogenin peptide (TRAP)), and Fraction A, containing a mixture of >6 kDa peptides (including a leucine-rich amelogenin peptide (LRAP))) differentially regulate osteogenic differentiation of periodontal ligament (PDL) cells. The present study examined whether EMD and the EMD Fractions (i) bind and internalize into PDL cells and (ii) precipitate and form insoluble complexes on PDL cells. MATERIALS AND METHODS: Biotin-labelled EMD/EMD Fractions were incubated with PDL cells under various different culture conditions and confocal and electron microscopies were carried out to examine the binding and intracellular trafficking of these proteins. RESULTS: The results reported here show, for the first time, that at least some components in Fraction A and the TRAP peptide in Fraction C can bind and be internalized by human PDL cells via receptor-mediated endocytosis. In addition, Fraction A was found to form insoluble aggregate-like structures on PDL cells, whereas Fraction C was soluble in culture media. CONCLUSION: Soluble amelogenin isoform TRAP appears to be internalizing into a subset of PDL cells. Moreover, TRAP uptake is most likely controlled by receptor-mediated endocytosis. CLINICAL RELEVANCE: Information on interaction between PDL cells and EMD/TRAP might prove useful in designing targeted interventions (i.e. use of chemically prepared soluble amelogenin peptides) to repair/regenerate periodontal tissues. Such interventions can also (i) avoid the use of rather crude animal-derived enamel matrix protein (EMP)/EMD preparation and (ii) preparation of cost-effective and more controlled chemically synthesized amelogenin peptides for the clinical use.

Treatment of intrabony defects with guided tissue regeneration in aggressive periodontitis: clinical outcomes at 6 and 12 months.

OBJECTIVE: The aim of this study was to compare clinical outcomes between guided tissue regeneration (GTR) and access flap (AF) surgery in patients with aggressive periodontitis (AgP). METHODS: Eighteen AgP patients with similar bilateral intrabony defects were treated in this split-mouth, single-blinded, randomised, controlled clinical trial. All patients presented with ≥3 mm intrabony defects and ≥5 mm periodontal pocket depths (PPD). In each patient, one defect was treated with a polyglycolide membrane according to the GTR principle, whereas the contralateral side was treated with AF. For both sides, a simplified papilla preservation flap was used. At baseline, 6 and 12 months post-surgery, the clinical attachment levels (CAL) and PPD were evaluated. RESULTS: At 6 and 12 months, at the GTR sites, the mean [95 % CI] CAL gain was 1.7 mm [1.1, 2.3] and 1.6 mm [0.9, 2.1], respectively, while the mean [95 % CI] PPD reduction was 2.3 mm [1.9, 2.8] and 2.4 mm [1.9, 2.8], respectively. Similar CAL (1.6 mm [1.0, 2.2] and 2.1 mm [1.4, 2.7]) and PPD (2.0 mm [1.5, 2.4] and 2.5 mm [2.0, 3.0]) outcomes were observed at the control sites at 6 and 12 months, respectively. Notably, at the GTR-treated sites, 13 subjects presented with various degrees of membrane exposure. CONCLUSIONS: Both therapies were effective in the treatment of intrabony defects in AgP patients, and no statistically significant differences between them could be demonstrated, possibly as a result of the differing degrees of membrane exposure at the GTR sites. CLINICAL RELEVANCE: Both periodontal regeneration and conventional periodontal surgery are effective treatments for AgP patients.

Radiographic outcomes following treatment of intrabony defect with guided tissue regeneration in aggressive periodontitis.

OBJECTIVES: This study reports the radiographic analysis of a split-mouth, single-blinded, randomised controlled clinical trial which was designed to compare the efficacy of simplified papilla preservation flap (SPPF) with or without guided tissue regeneration (GTR) in patients with aggressive periodontitis (AgP). METHODS: Eighteen AgP patients who had similar bilateral intrabony defects were treated. In all patients, the defects presented with radiographic evidence of an intrabony defect ≥3 and ≥5 mm of periodontal pocket depths (PPD). The surgical procedures included access for root instrumentation using SPPF alone (control) or, after debridement, a placement of resorbable GTR membrane (test). The standardised radiographic assessments were carried out at pre-surgical baseline and at 6 and 12 month post-surgery. Radiographic linear measurements and subtraction radiography were used as the method of analysis. RESULTS: Both treatments showed significant improvements in linear radiographic bone fill and defect resolution at 6 and 12 months, compared to baseline. The 12-month subtraction radiography at the GTR sites showed a significant improvement compared to the 6-month outcomes. CONCLUSIONS: Both therapies were effective in the treatment of intrabony defects in AgP patients although no significant differences between them could be demonstrated. The finding that the bone fill and resolution of the defect at the GTR sites were significantly higher at 12 months than at 6 months after treatment indicates that bone regeneration is still an ongoing process at 6 months post-surgery. CLINICAL RELEVANCE: Radiographic assessment of periodontal regeneration should be carried out at 12 months post-surgery in order to evaluate the complete healing of the bony defect.

A procedure for identifying stem cell compartments with multi-lineage differentiation potential.

Stem cells isolated from adult human tissue have received increasing attention because of their potential to repair and/or regenerate damaged tissue. However, identification and characterization of such cell populations have been limited due to the lack of adequate methodology for assessing their multi-lineage potential. In the present study, using adult human ligament tissue as a model, we have developed a combination of methods which together can be used to identify adult stem cell compartments based on their ability to undergo a range of differentiation pathways, including osteogenesis, adipogenesis, chondrogenesis, myogenesis, vasculogenesis, angiogenesis, neurogenesis and gliogenesis in vitro. This was carried out using the conventional reverse transcription polymerase chain reaction technique to assess the expression of selected key lineage-associated marker genes and by using histological, immunological and morphological criteria to assess characteristic features of lineage-specific 'terminal' differentiation in vitro.

Reactive calcium-phosphate-containing poly(ester-co-ether) methacrylate bone adhesives: setting, degradation and drug release considerations.

This study has investigated novel bone adhesives consisting of fluid photo-polymerizable poly(lactide-co-propylene glycol-co-lactide)dimethacrylate (PGLA-DMA) mixed with systematically varying fillers of ß-tricalcium phosphate (ß-TCP) and monocalcium phosphate monohydrate (MCPM), for the delivery of an antibacterial drug chlorhexidine (CHX). All formulations were found to polymerize fully within 200 s after exposure to blue light. In addition, water sorption by the polymerized materials catalyzed varying filler conversion to dicalcium phosphate (DCP) (i.e. brushite and monetite). With greater DCP levels, faster degradation was observed. Moreover, increase in total filler content enhanced CHX release, associated with higher antibacterial activity. These findings thus suggest that such rapid-setting and degradable adhesives with controllable drug delivery property could have potential clinical value as bone adhesives with antibacterial activity.

Development and validation of a multiplex bead assay for measuring growth mediators in wound fluid.

Large amounts of biological samples are usually required to measure multiple components by the enzyme-linked immunosorbant assay. However, the amounts of many tissue extracts and fluids, including gingival crevicular fluid (GCF), are generally extremely small. The aim of this study was, therefore, to develop and validate a novel multiplex bead assay (MBA) to simultaneously measure a profile of healing-related mediators in the GCF of treated periodontal wounds. An MBA was developed and validated by assessment of assay selectivity, recovery, precision and sensitivity, using eight recombinant human growth mediators as assay standards. GCF samples were collected on paper strips from healing wound (test) and healthy unaffected (control) sites of 15 patients with periodontitis, seven days post-periodontal surgery. Each GCF sample was eluted and the levels of the mediators measured using the MBA and antibody pairs specific for angiopoietin-1, vascular endothelial growth-factor, bone morphogenetic protein-2, osteoprotegerin, tissue inhibitor of metalloprotease-1 (TIMP-1), basic fibroblast growth-factor, keratinocyte growth-factor, and platelet derived growth-factor. Less than 1.8% of cross-reactivity was observed between antibodies and the eight different analytes, for which the recovery was more than 85%. Mean intra- and inter-assay precision were within the acceptance criteria of 20% and 25%, respectively. Detection of all mediators was highly sensitive (

Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2.

Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor beta1 (TGF-beta1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-beta1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-beta1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.

Modulation of the human bone cell cycle by calcium ion-implantation of titanium.

Ca ion implantation of Ti surfaces has previously been reported to enhances osseointegration in vivo. Although the mechanisms underlying the response of bone cells to these novel surfaces still remain unclear, it is possible that Ca ion-implanted Ti (Ca-Ti) may influence the growth of new bone by modulating the progression of the cell cycle. In the present study we have, therefore, examined the precise effects of Ca ion-implantation of Ti on the bone-like MG-63 cell line in vitro. The results of flow cytometry analysis showed that this surface markedly enhanced the proportion of cells which expressed Ki-67, a cell proliferation-associated nuclear antigen, compared with cells grown on the non-implanted Ti (control) surface. In addition, cultures grown on Ca-Ti and synchronized at the G1/S boundary by hydroxyurea more rapidly re-entered and progressed through the S and G2/M phases of the cell cycle than their counterparts on Ti. Ca ion-implantation also significantly increased the numbers of mitotic cells. These results thus show that alteration of the surface chemistry of Ti by high-energy implantation with Ca ion was able to substantially modulate the progression of the bone cell cycle, and suggest a possible means of enhancing the response of bone cells to implant materials.

Effects of calcium ion-implantation of titanium on bone cell function in vitro.

The modification of titanium (Ti) surfaces by ion-implantation has previously been reported to enhance osseointegration in vivo. However, the mechanisms underlying the apparently improved biocompatibility of these novel implant materials are unknown. The aim of this study is, therefore, to determine the precise effects of calcium ion-implanted Ti on the functional activity of bone cells in vitro. Flow cytometry (FCM) and the reverse transcriptase polymerase chain reaction (RT-PCR) were used to measure the response of bone-derived cells to key bone-associated components, including alkaline phosphatase (ALP), bone morphogenetic protein receptor-1B (BMPR-1B), bone sialoprotein (BSP), osteonectin (ON), and osteopontin (OPN). FCM analysis showed that BMPR-1B, BSP and particularly OPN were significantly up-regulated in MG-63 cells cultured on Ca-implanted Ti compared with control nonimplanted Ti. Moreover, the effects of this novel Ca-Ti surface were found to be mediated, at least partly, via gene activation, since RT-PCR demonstrated the presence of notably elevated levels of OPN mRNA transcripts in the MG-63 cells. These findings thus show that Ti surfaces implanted with Ca ions can enhance the expression of certain bone-associated components in vitro, and suggest that this effect could be the cause of the potential benefit of this material on bone in vivo.

Effects of calcium ion implantation on human bone cell interaction with titanium.

The use of calcium ion (Ca) implantation of titanium (Ti), previously reported to encourage osseointegration in vivo, has been investigated using an in vitro model in order to understand the basic mechanisms involved in the response of target cells to such surfaces. Polished Ti discs were implanted with high, medium and low (1x10(17), 1x10(16), 1x10(15)ionscm-2) doses of Ca ions at 40 keV. The effects of different levels of Ca implantation on morphology, attachment and spreading of MG-63 cells seeded on the surface of control (non-implanted) Ti and Ca-Ti discs were assessed. Further, to understand cell-material interactions at a molecular level, the expression of beta1 and alpha5beta1 integrins and the formation of vinculin-positive focal adhesion plaques were examined. In addition, the effects of pre-immersion of the Ca (high)-Ti in tissue culture medium on cell attachment were measured and correlated with specific chemical changes at the Ti surface. Our findings suggest that Ca implantation can affect the adhesion of MG-63 cells both qualitatively and quantitatively. However, this effect appears to depend on the level at which Ca ions are implanted. Results showed that although cell adhesion on Ca (high)-Ti was initially reduced, it nevertheless was not only restored but substantially increased with progressing culture times. In addition, a significantly enhanced cell spreading, formation of focal adhesion plaques and expression of integrins were measured on this particular surface. In contrast, no marked differences were observed in cell behaviour on Ca-Ti (low and medium). Pre-immersion studies indicated that the decrease in cell attachment to Ca (high)-Ti at early time periods may be linked to the presence of Ca- and P-rich particles on the surface. The absence of these particles at 24 h was consistent with a significant increase in cell attachment.

Changes in soluble adhesion molecules in gingival crevicular fluid following periodontal surgery.

BACKGROUND: Inflammation of periodontal tissues during postoperative wound healing is mediated by cell surface adhesion molecules. Soluble forms of these antigens have also been identified and shown to be important in immunoregulatory processes, but have previously not been investigated during periodontal repair and regeneration. The present study has examined the presence and possible changes in soluble intercellular adhesion molecule-1 (sICAM-1; CD54) and lymphocyte function-associated antigen-3 (sLFA-3; CD58) in gingival crevical fluid (GCF) following periodontal surgery. METHODS: GCF samples were collected from four groups: 1) a guided tissue regeneration (GTR) test; 2) a GTR control, at least one complete tooth unit away from the periodontal defect; 3) a conventional flap (CF) surgery; and 4) a crown lengthening (CL). Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of sICAM-1 and sLFA-3 in the GCF samples. RESULTS: A marked increase in GCF volumes was found in all sites after surgery, although a persistent increase was associated only with the period of membrane retention at the GTR test sites. In addition, sICAM-1 and sLFA-3 were found in the GCF of healthy as well as diseased sites prior to treatment and the total amounts of both increased transiently following surgical intervention, especially sLFA-3. However, the concentrations of these GCF components, particularly sICAM-1, tended to decrease. CONCLUSIONS: The temporal decrease in the concentration of sICAM-1 and sLFA-3 in GCF may serve to enhance inflammatory reactions at surgically-treated periodontal sites, thereby limiting repair and regeneration in the periodontium. These soluble adhesion molecules may thereby be of potential therapeutic value and might also be useful markers for monitoring periodontal wound healing.

A tyrosine-rich amelogenin peptide promotes neovasculogenesis in vitro and ex vivo.

The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo.

Effects of enamel matrix proteins on multi-lineage differentiation of periodontal ligament cells in vitro.

The adult periodontal ligament (PDL) is considered to contain progenitor cells that are involved in the healing of periodontal wounds. Treatment with enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs), has been shown to be of some clinical benefit in eliciting periodontal regeneration in vivo. Although there is extensive information available about the effects of EMD on periodontal regeneration, the precise influence of this material on alveolar bone and the formation of blood vessels and proprioceptive sensory nerves, prominent features of functionally active periodontal tissue, remain unclear. The aim of the present study was therefore to examine the effects of EMD on the ability of human periodontal ligament cells (HPCs) to undergo multi-lineage differentiation in vitro. Our results showed that HPCs treated with EMD under non-selective growth conditions did not show any evidence of osteogenic, adipogenic, chondrogenic, neovasculogenic, neurogenic and gliogenic "terminal" differentiation. In contrast, under selective lineage-specific culture conditions, EMD up-regulated osteogenic, chondrogenic and neovasculogenic genes and "terminal" differentiation, but suppressed adipogenesis, neurogenesis and gliogenesis. These findings thus demonstrate for the first time that EMD can differentially modulate the multi-lineage differentiation of HPCs in vitro.

Differential effect of amelogenin peptides on osteogenic differentiation in vitro: identification of possible new drugs for bone repair and regeneration.

Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases.

Endogenous BMPR-IB signaling is required for early osteoblast differentiation of human bone cells.

Osteoblast differentiation is tightly regulated by a number of cytokines and growth factors, including bone morphogenetic proteins (BMP) which stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II). Although the mechanisms which regulate osteoblast differentiation are not fully understood, it is possible that endogenous BMPR signaling could play an important part in this process. To test this hypothesis, we have examined the expression and the functional significance of BMPR during osteoblast differentiation of primary human bone cells. The results showed that although the expression of BMPR-IA and -II transcripts were constantly expressed while the bone cells underwent osteoblast differentiation, the level of BMPR-IB mRNA was transiently, but significantly, up-regulated by threefold on day 3. This increase in BMPR-IB expression was found to be associated with the significant up-regulation of core binding factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) transcripts as well as the ALP activity, the well-established early markers of osteoblast differentiation. Transfection of bone cells with BMPR-IB small interfering RNA (siRNA) was found to significantly ablate the expression of BMPR-IB which subsequently resulted in reduction of Cbfa1 and ALP mRNA as well as the ALP activity. Moreover, exogenously added BMP-2 failed to rescue osteoblast differentiation of BMPR-IB siRNA-transfected bone cells. In conclusion, the present study has shown that endogenous BMPR-IB signaling is required for early phase of osteoblast differentiation of human bone cells in vitro, suggesting that BMPR-IB could be a therapeutic target for initiating bone healing in vivo.

Reactive calcium-phosphate-containing poly(ester-co-ether) methacrylate bone adhesives: chemical, mechanical and biological considerations.

A poly(propylene glycol-co-lactide) dimethacrylate adhesive with monocalcium phosphate monohydrate (MCPM)/beta-tricalcium phosphate (beta-TCP) fillers in various levels has been investigated. Water sorption by the photo-polymerized materials catalyzed varying filler conversion to dicalcium phosphate (DCP). Polymer modulus was found to be enhanced upon raising total calcium phosphate content. With greater DCP levels, faster release of phosphate and calcium ions and improved buffering of polymer degradation products were observed. This could reduce the likelihood of pH-catalyzed bulk degradation and localized acid production and thereby may prevent adverse biological responses. Bone-like MG-63 cells were found to attach, spread and have normal morphology on both the polymer and composite surfaces. Moreover, composites implanted into chick embryo femurs became closely apposed to the host tissue and did not appear to induce adverse immunological reaction. The above results suggest that the new composite materials hold promise as clinical effective bone adhesives.

Isolation and characterization of stem cell clones from adult human ligament.

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics and to play an important part in re-building damaged tissue, including alveolar bone. However, these populations have been heterogeneous, and thus far no highly purified periodontal stem cell (PSC) clone has yet been established from adult human PDL tissue. The present study was therefore carried out to isolate single cell-derived PDL clones and to delineate their phenotypic and functional characteristics. In this report we have obtained four homogeneous and distinct clones--namely, C5, C6, C7, and C8--and have found these to be highly proliferative and to express the stromal cell markers CD29 and CD44. In particular, C7 showed stem cell-like characteristics of small cell size with reduced cytoplasm, clonogenicity, and multilineage potential, including osteogenic activity in forming bone-like tissue in organoid micromass cultures. Clones C5 and C6 possessed osteoprogenitor features with mineralized matrix-forming activity, whereas C8 did not undergo osteogenic, adipogenic, or chondrogenic differentiation. The present study thus reports, for the first time, the isolation and cellular and molecular characterization of highly purified putative PSC and osteoprogenitors in adult human PDL, based on clonogenicity and multilineage differentiation potential, with PSC-C7 capable of bone formation in vitro, suggesting that such cells may have potential value for stem cell-based bone tissue engineering in vivo.

Up-regulation of bone morphogenetic protein receptor IB by growth factors enhances BMP-2-induced human bone cell functions.

Bone morphogenetic proteins (BMP) stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB, and -II). Several growth factors, including transforming growth factor-beta1 (TGF-beta1), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-AB (PDGF-AB), have also been shown to play an important part in osteogenesis. The mechanism underlying these activities is unclear, but these growth factors could modulate the BMP/BMPR pathway by up-regulating BMPR expression, thereby enhancing the osteogenic responses of bone cells to the BMP. In this study we have therefore examined the effects of TGF-beta1, FGF-2, and PDGF-AB on BMPR expression and BMP-2-mediated osteoblast functions in primary human bone cells. The results showed that although the ligand BMP-2 and growth factors had little effect on BMPR-IA and -II transcript expression, they significantly up-regulated BMPR-IB mRNA specifically. However, only the growth factors, but not the ligand BMP-2, increased the surface expression of the BMPR-IB antigen, which was found to be due to a differential effect of BMP-2 and the growth factors on the Smurf1/Smad6-induced breakdown process. Pre-incubation of the cells with the growth factors significantly augmented BMP-2-induced Smad1/5/8 phosphorylation, and Dlx5 expression ALP activity, compared with that of cells treated with BMP-2 alone. When cells were transfected with siRNA targeting BMPR-IB, the growth factors neither up-regulated BMPR-IB transcript expression nor enhanced BMP-2-induced Smad1/5/8 phosphorylation, Dlx5 expression and ALP activity. The results indicate that increased BMPR-IB by TGF-beta1, FGF-2, and PDGF-AB significantly enhances BMP-2-induced osteogenic functions in vitro, suggesting that they might positively modulate bone formation by up-regulating BMPR-IB in vivo.

Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.