Dual-Functional Drug Delivery System for Bisphosphonate-Related Osteonecrosis Prevention and Its Bioinspired Releasing Model and In Vitro Assessment.

Clindamycin (CDM)/geranylgeraniol (GGOH)-loaded plasma-treated mesoporous silica nanoparticles/carboxymethyl chitosan composite hydrogels (CHG60 and CHG120) were developed for the prevention of medication-related osteonecrosis of the jaw associated with bisphosphonates (MRONJ-B). The pore structure and performances of CHGs, e.g., drug release profiles and kinetics, antibacterial activity, zoledronic acid (ZA)-induced cytotoxicity reversal activity, and acute cytotoxicity, were evaluated. The bioinspired platform mimicking in vivo fibrin matrices was also proposed for the in vitro/in vivo correlation. CHG120 was further encapsulated in the human-derived fibrin, generating FCHG120. The SEM and µCT images revealed the interconnected porous structures of CHG120 in both pure and fibrin-surrounding hydrogels with %porosity of 75 and 36%, respectively, indicating the presence of fibrin inside the hydrogel pores, besides its peripheral region, which was evidenced by confocal microscopy. The co-presence of GGOH moderately decelerated the overall releases of CDM from CHGs in the studied releasing fluids, i.e., phosphate buffer saline-based fluid (PBB) and simulated interstitial fluid (SIF). The whole-lifetime release patterns of CDM, fitted by the Ritger-Peppas equation, appeared nondifferentiable, divided into two releasing stages, i.e., rapid and steady releasing stages, whereas the biphasic drug release patterns of GGOH were observed with Phase I and II releases fitted by the Higuchi and Ritger-Peppas equations, respectively. Notably, the burst releases of both drugs were subsided with lengthier durations (up to 10-12 days) in SIF, compared with those in PBB, enabling CHGs to elicit satisfactory antibacterial and ZA cytotoxicity reversal activities for MRONJ-B prevention. The fibrin network in FCHG120 further reduced and sustained the drug releases for at least 14 days, lengthening bactericidal and ZA cytotoxicity reversal activities of FCHG and decreasing in vitro and in ovo acute drug toxicity. This highlighted the significance of fibrin matrices as appropriate in vivo-like platforms to evaluate the performance of an implant.

Finite Element Analysis of the Mechanical Performance of Non-Restorable Crownless Primary Molars Restored with Intracoronal Core-Supported Crowns: A Proposed Treatment Alternative to Extraction for Severe Early Childhood Caries.

Early childhood caries (ECC) involve extensive coronal tooth structure loss, and tooth reconstruction remains highly challenging. To fulfill preclinical assessment, the present study investigated the biomechanics of non-restorable crownless primary molars that were restored by stainless steel crowns (SSC) using different composite core build-up materials. Computer-aided design-integrated 3D finite element and modified Goodman fatigue analyses were performed to determine stress distribution, risk of failure, fatigue life and dentine-material interfacial strength for the restored crownless primary molars. A dual-cured resin composite (MultiCore Flow), a light-cured bulk-fill resin composite (Filtek Bulk Fill posterior), a resin-modified glass-ionomer cement (Fuji II LC) and a nano-filled resin-modified glass-ionomer cement (NRMGIC; Ketac N100) were used as core build-up composite materials in the simulated models. The finite element analysis showed that types of core build-up materials affected the maximum von Mises stress only in the core materials (p-value = 0.0339). NRMGIC demonstrated the lowest von Mises stresses and revealed the highest minimum safety factor. The weakest sites were along the central grooves regardless of type of material, and the ratio of shear bond strength to maximum shear stress at the core-dentine interface of the NRMGIC group was lowest among the tested composite cores. However, all groups provided lifetime longevity from the fatigue analysis. In conclusion, core build-up materials differentially influenced the von Mises stress (magnitude and distribution) and the safety factor in crownless primary molars restored with core-supported SSC. However, all materials and the remaining dentine of crownless primary molars provided lifetime longevity. The reconstruction by core-supported SSC, as an alternative to tooth extraction, may successfully restore non-restorable crownless primary molars without unfavorable failures throughout their lifespan. Further clinical studies are required to evaluate the clinical performance and suitability of this proposed method.

Biodegradable Dual-Function Nanocomposite Hydrogels for Prevention of Bisphosphonate-Related Osteonecrosis of the Jaw.

This study presents the development of composite hydrogels, comprising a biodegradable polymer (carboxymethyl chitosan (CMCS or CM)) and a mixture of plasma-treated mesoporous silica nanoparticles (PMCM-41 or PM) and amine-functionalized mesoporous silica nanoparticles (NMCM-41 or NM), coloaded with a hydrophilic antibiotic (clindamycin hydrochloride (CDM or C)) and a poorly water-soluble compound (geranylgeraniol (GGOH or G)) for prevention of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The CG-loaded hydrogel stabilities were better maintained when CDM-preloaded PMCM-41 and NMCM-41 were initially used and governed by weight ratios of CDM-loaded PMCM-41 to NMCM-41 and CDM quantity utilized. 5PM240C-1NM-CM demonstrated the best CDM-loaded hydrogel for GGOH postloading. The scanning electron microscopy (SEM) and X-ray microcomputer-tomography (µCT) images of 5PM240C-1NM-CM revealed a porous structure with homogeneously distributed nanoparticles. Two GGOH-loaded 5PM240C-1NM-CM hydrogels were generated after GGOH loadings. Their biphasic drug release profiles were fitted by Ritger-Peppas and Hixson-Crowell models. The copresence of GGOH could hinder CDM releases, while GGOH was released with a slower rate. The hydrogels prolonged the CDM and GGOH releases up to 9 days. They possessed antibacterial activities against Streptococcus sanguinis for up to 14 days and satisfactorily provided good cytoprotection against zoledronic acid for osteoclastic and osteoblastic progenitors, thus preserving a pool of viable progenitor cells that had the capacity to differentiate into mature osteoclasts and osteoblasts in vitro, suggesting their potential local application for prevention of BRONJ.

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.

IFNγ-primed periodontal ligament cells regulate T-cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact.

Periodontal ligament (PDL) cells help maintain tissue homeostasis by balancing PDL tissue inflammation and regeneration. However, the mechanisms by which interferon γ (IFNγ) modulate this process are not yet fully understood. The present study aimed to examine the effect of primed and non-primed PDL cells with IFNγ on the viability and differentiation of T lymphocytes and its functional consequences. The results showed that IFNγ-primed PDL cells possessed enhanced immunosuppression by suppressing T-lymphocyte viability and directing T-lymphocyte differentiation towards a higher T helper (Th) Th2/Th1 ratio. Suppression of T-cell viability was mainly mediated by IFNγ-inducible secreted mediators, which was prevented in the presence of direct cell contact, probably by intercellular adhesion molecule-1 (ICAM-1)-induced PI3 K-mediated transforming growth factor ß1 expression in PDL cells. By contrast, ICAM-1 activation augmented IFNγ-induced IFNγ and interleukin-6 expression in PDL cells, which in turn modulated T-cell differentiation. The resulting interaction between these two cell types activated macrophage and suppressed osteoclast differentiation. In conclusion, the results have shown, for the first time to our knowledge, that primed and non-primed PDL cells with IFNγ differentially control T-cell responses via IFNγ-inducible mediators and ICAM-1-mediated direct cell contact, suggesting the role of PDL cells in shifting an inflammatory phase towards a regenerative phase.

Antibacterial and osteogenic activities of clindamycin-releasing mesoporous silica/carboxymethyl chitosan composite hydrogels.

Conventional treatment of jaw bone infection is often ineffective at controlling bacterial infection and enhancing bone regeneration. Biodegradable composite hydrogels comprised of carboxymethyl chitosan (CMCS) and clindamycin (CDM)-loaded mesoporous silica nanoparticles (MCM-41), possessing dual antibacterial activity and osteogenic potency, were developed in the present study. CDM was successfully loaded into both untreated and plasma-treated MCM-41 nanoparticles, denoted as (p)-MCM-41, followed by the incorporation of each of CDM-loaded (p)-MCM-41 into CMCS. The resulting CDM-loaded composite hydrogels, (p)-MCM-41-CDM-CMCS, demonstrated slow degradation rates (about 70% remaining weight after 14-day immersion), while the CDM-free composite hydrogel entirely disintegrated after 4-day immersion. The plasma treatment was found to improve drug loading capacity and slow down initial drug burst effect. The prolonged releases of CDM from both (p)-MCM-41-CDM-CMCS retained their antibacterial effect against Streptococcus sanguinis for at least 14 days in vitro. In vitro assessment of osteogenic activity showed that the CDM-incorporated composite hydrogel was cytocompatible to human mesenchymal stem cells (hMSCs) and induced hMSC mineralization via p38-dependent upregulated alkaline phosphatase activity. In conclusion, novel (p)-MCM-41-CDM-CMCS hydrogels with combined controlled release of CDM and osteogenic potency were successfully developed for the first time, suggesting their potential clinical benefit for treatment of intraoral bone infection.

Geranylgeraniol prevents zoledronic acid-mediated reduction of viable mesenchymal stem cells via induction of Rho-dependent YAP activation.

Long-term use of zoledronic acid (ZA) increases the risk of medication-related osteonecrosis of the jaw (MRONJ). This may be attributed to ZA-mediated reduction of viable mesenchymal stem cells (MSCs). ZA inhibits protein geranylgeranylation, thus suppressing cell viability and proliferation. Geranylgeraniol (GGOH), which is a naturally found intermediate compound in the mevalonate pathway, has positive effects against ZA. However, precise mechanisms by which GGOH may help preserve stem cell viability against ZA are not fully understood. The objective of this study was to investigate the cytoprotective mechanisms of GGOH against ZA. The results showed that while ZA dramatically decreased the number of viable MSCs, GGOH prevented this negative effect. GGOH-rescued ZA-exposed MSCs formed mineralization comparable to that produced by normal MSCs. Mechanistically, GGOH preserved the number of viable MSCs by its reversal of ZA-mediated Ki67+ MSC number reduction, cell cycle arrest and apoptosis. Moreover, GGOH prevented ZA-suppressed RhoA activity and YAP activation. The results also established the involvement of Rho-dependent YAP and YAP-mediated CDK6 in the cytoprotective ability of GGOH against ZA. In conclusion, GGOH preserves a pool of viable MSCs with osteogenic potency against ZA by rescuing the activity of Rho-dependent YAP activation, suggesting GGOH as a promising agent and YAP as a potential therapeutic target for MRONJ.

Titanium dioxide nanotubes of defined diameter enhance mesenchymal stem cell proliferation via JNK- and ERK-dependent up-regulation of fibroblast growth factor-2 by T lymphocytes.

Long-term clinical success of a titanium implant not only depends upon osseointegration between implant and bone surface but also on the response of host immune cells. Following implantation of biomaterials, an inflammatory response, including T lymphocyte response, is ostensibly initiated by implant-cell interaction. However, little is known about the responses of T lymphocytes to titanium dioxide nanotubes. The present study aimed to explore the effect of titanium dioxide nanotubes on T lymphocytes in vitro and its biological consequences. The results of the present study showed that titanium dioxide nanotubes with diameter of 30-105 nm were non-cytotoxic to T lymphocytes, and the 105 nm titanium dioxide nanotube surface specifically possessed an ability to activate T lymphocytes, thus increasing DNA synthesis and cell proliferation. In addition, the 105 nm titanium dioxide nanotubes significantly activated the expression of FGF-2 gene and protein in T lymphocytes although smaller nanotubes (i.e. those with diameters of approximately 30 and 70 nm) had little effect on this. The present study investigated the mechanism by which 105 nm nanotubes stimulated FGF-2 expression in T lymphocytes by blocking key MAPK pathways. The inhibitors of JNK1/2/3 and ERK1/2 significantly inhibited 105 nm titanium dioxide nanotubes-induced FGF-2 expression. Corresponding to the increased expression of FGF-2, only the supernatant from T lymphocytes cultured on 105 nm nanotubes stimulated human mesenchymal stem cell proliferation. FGF-2 blocking antibody partially reversed the increased proliferation of human mesenchymal stem cells, supporting the role of T lymphocyte-derived FGF-2 in enhanced human mesenchymal stem cell proliferation. This suggests a significant role of T lymphocyte-titanium dioxide nanotube interaction in the proliferation of human mesenchymal stem cells, which is pivotal to the formation of new bone following implant placement.

Osteoinduction of stem cells by collagen peptide-immobilized hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate scaffold for bone tissue engineering.

Bone substitute is a therapeutic approach to treat bone abnormalities. A scaffold serves mainly as osteoconductive elements. To facilitate a better biological performance, short collagen peptide was immobilized onto hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate (HPBSu/TCP) scaffolds. PBSu/TCP (80:20) scaffolds were fabricated by a supercritical CO2 technique, hydrolyzed with 0.6 M NaOH and conjugated with short collagen peptide tagged with or without red fluorescence. The surface morphology and porous structure of scaffolds were characterized by scanning electron microscopy and micro-computed tomography. Human mesenchymal stem cells were cultured onto the scaffolds and examined for osteogenic differentiation and biomineralization in vitro by means of alkaline phosphatase activity, alizarin red staining, and reverse transcription-polymerase chain reaction. The PBSu/TCP and HPBSu/TCP scaffolds were successfully prepared. Scanning electron microscopy and micro-computed tomography results showed that the porosity was distributed throughout the scaffolds with the pore sizes in the range of 250-900 µm. Fluorescence microscopy demonstrated retention of tagged short collagen peptide on the scaffold. Mesenchymal stem cells adhered and grew well on the material. Under osteogenic induction, cells cultured on the short collagen peptide -immobilized scaffold significantly produced a greater amount of alkaline phosphatase activity and positive mineralization than those cultured on the control scaffold. The present results have shown that the short collagen peptide-immobilized HPBSu/TCP scaffold enhanced osteoinduction and biomineralization of stem cell-derived osteoblasts, possibly via stimulation of alkaline phosphatase activity. This suggests the potential use of osteogenic peptide-immobilized material in bone tissue engineering for correcting bone defects.

In-vitro responses of T lymphocytes to poly(butylene succinate) based biomaterials.

BACKGROUND: Polybutylene succinate (PBSu) and PBSu/ß-tricalcium phosphate (TCP) composites are biocompatible and good candidates as bone graft materials. However, little is known about the responses of T lymphocytes to these biomaterials, which play an important role in the success of bone grafting. METHODS: Activated T lymphocytes were cultured onto 32 mm diameter films (PBSu/TCP films), that had previously been placed in 6-well culture plates, for 8, 24 and 72 hours. A plastic-well culture plate was used as a control surface. The effects of PBSu-based biomaterials on T lymphocytes were examined by the using flow cytometry and reverse-transcription polymerase chain reaction. RESULTS: These biomaterials were non-toxic to T lymphocytes, allowing their normal DNA synthesis and activation. All materials induced only transient activation of T lymphocytes, which existed no longer than 72 hours. Proportions of four main CD4/CD8 T lymphocyte subpopulations were not affected by these biomaterials. Moreover, PBSu and PBSu/TCP significantly suppressed the expression of IL-1ß and IL-6 genes by 15-35% and 21-26%, respectively. In contrast, a PBSu/TCP composite (at PBSu:TCP=60:40) significantly stimulated the expression of IL-10 and IL-13 genes by 17% and 19%, respectively. CONCLUSIONS: PBSu and PBSu/TCP composites were non-toxic to T lymphocytes and did not induce unfavorable responses of T lymphocytes. The tested biomaterials down-regulated key proinflammatory cytokine genes and up-regulated anti-inflammatory cytokine genes in T lymphocytes. These suggest that the biomaterials studied are good candidates as bone graft materials.

Effect of bidirectional loading on contact and force characteristics under a newly developed masticatory simulator with a dual-direction loading system.

Mechanical responses of the test specimen under bidirectional and unidirectional loading were investigated using a newly developed masticatory simulator. The simulator adopted a four-bar linkage mechanism to create both loading patterns. Scratch/occlusal contact characteristics, contact force profiles and the fracture of restored tooth samples were investigated. With bidirectional loading, which imitates the nature of human chewing cycle closer than the unidirectional loading does, the occlusal contact was ovoid in shape whereas a small circular area was observed from the test with unidirectional loading. The contact force profiles were also noticeably dependent on the loading patterns. Measured contact forces from bidirectional loading were more uniform than those from unidirectional loading. Bidirectional loading also induced the cuspal fracture with similar characteristics of natural cuspal fractures in humans. The differences of force characteristics between those of bidirectional and unidirectional loadings emphasize the importance of employing bidirectional loading in dental material testing.

Stem cell adhesion and proliferation on hydrolyzed poly(butylene succinate)/ß-tricalcium phosphate composites.

Although poly(butylene succinate)/ß-tricalcium phosphate (PBSu/TCP) composites are biocompatible and allow the growth and osteogenic differentiation of stem cells, cell attachment and adhesion to the PBSu-based substrates is often limited. To enhance cell adhesion and proliferation, we used a sodium hydroxide (NaOH) hydrolysis technique to generate a different degree of roughness on PBSu/TCP substrates with different PBSu:TCP ratios. The results showed that NaOH hydrolysis increased surface roughness of PBSu/TCP substrates in a concentration-dependent manner. Substrates with higher ratios of TCP:PBSu provided more porous topography after NaOH hydrolysis, with a substrate containing 40 wt % TCP (PBSu/TCP-6040) hydrolyzed with 1.5M NaOH (HPBSu/TCP-6040-1.5) showing the highest degree of roughness. As with the roughness, PBSu/TCP surface hydrophilicity was positively affected by the increasing NaOH concentration and TCP incorporation. Stem cells adhered best on HPBSu/TCP-6040-1.5 with three-dimensionally elongated cell extensions. Moreover, the HPBSu/TCP-6040-1.5 substrate most significantly facilitated stem cell actin cytoskeleton reorganization and vinculin-positive focal adhesion formation when compared with the other substrates tested. HPBSu/TCP-6040-1.5 also demonstrated the greatest increase in cell proliferation when compared with the other substrates studied. In conclusion, the results have shown that among various substrates tested, HPBSu/TCP-6040-1.5 provided the best support for stem cell adhesion and proliferation, suggesting its potential use in bone engineering.

Enhanced osteogenic activity of a poly(butylene succinate)/calcium phosphate composite by simple alkaline hydrolysis.

Bone engineering offers the prospect of alternative therapies for clinically relevant skeletal defects. Poly(butylene succinate) (PBSu) is a biodegradable and biocompatible polyester which may possess some limitations in clinical use due to its hydrophobicity. In order to overcome these limitations and increase the bioactivity, a simple and convenient surface hydrolysis of PBSu, PBSu/hydroxyapatite and PBSu/ß-tricalcium phosphate (TCP) films was performed. The resulting surfaces (i.e., HPBSu, HPBSu/HA and HPBSu/TCP) were tested for their physicochemical property, biocompatibility and osteogenic potency. The results showed that surface hydrolysis significantly increased surface roughness and hydrophilicity of the composites, with the HPBSu/TCP possessing the most pronounced results. All the materials appeared to be biocompatible and supported in vitro growth and osteoblast differentiation of hMSCs, and the alkaline hydrolysis significantly enhanced the hMSC cell proliferation and the osteogenic potency of PBSu/TCP compared with the non-hydrolyzed sample. In conclusion, the HPBSu/TCP possessed better hydrophilicity, biocompatibility and osteogenic potency in vitro, suggesting that this simple and convenient alkaline hydrolysis could be used to augment the biological property of PBSu-based composites for bone engineering in vivo.

Osteogenic potency of a 3-dimensional scaffold-free bonelike sphere of periodontal ligament stem cells in vitro.

OBJECTIVE: The present study aimed to investigate the osteogenic potency of scaffold-free 3-dimensional (3D) spheres of periodontal ligament stem cells (PDLSCs). STUDY DESIGN: The osteogenic potency of PDLSC spheres was determined by the ability to form mineralization and to express key osteogenesis-associated genes. The alkaline phosphatase (ALP) activity and the protein content of PDLSC spheres were also measured. RESULTS: The 3D sphere developed its osteogenic potency in a time-dependent manner, containing approximately 10-fold higher mineralization, 5-fold higher protein content, and 4-fold greater ALP activity than those in the controls. The expression of key osteogenic genes was also upregulated in the 3D PDLSC spheres. Cellular outgrowth was observed when reintroduced into 2D culture. CONCLUSIONS: PDLSCs were able to undergo osteogenic differentiation in a scaffold-free 3D culture, producing bonelike mineralization in vitro. This suggests, at least in vitro, the osteogenic potency of the 3D PDLSC spheres.

Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition.

BACKGROUND: Zoledronic acid (ZA) is prescribed to treat various metabolic bone diseases. Despite its efficacy in preventing bone loss, ZA has been linked to osteonecrosis of the jaw in several reports. However, a mechanism underlying this occurrence is still unclear. OBJECTIVE: This study was to investigate causative roles of ZA on osseous cellular activities of pre-osteoblastic cell line MC3T3-E1 (MC3T3) and mesenchymal stem cell (MSC). METHODS: Morphological analysis, RT-PCR, annexin V/PI staining, together with mineralization, cell viability, and alkaline phosphatase (ALP) activity assays were performed. RESULTS: Zoledronic acid treatment decreased bone nodule formation at all concentrations tested (0.01-100 µM). Cell morphologies of both cell types were altered from their normal appearances after the addition of ZA (≥ 5 µM), and cell viability was significantly inhibited at concentrations ≥ 0.1 µM for MC3T3 and at concentrations ≥ 10 µM for MSC. ZA (100 µM) induced apoptosis in MC3T3 and MSC. Furthermore, ALP activity from both cells was strongly reduced when exposed to ZA (≥ 1 µM for MC3T3 and ≥ 5 µM for MSC). ZA also down-regulated Runx 2 and Col I mRNA expressions. CONCLUSION: With this in vitro study, ZA mediated defective bone mineralization by directly disrupting osteoblast/osteoprogenitor cellular activities at several levels, that is, cell proliferation, osteoblast differentiation, and osteoblast function of both pre-osteoblastic cells and MSC.

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT.

Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.

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.

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.