Potentiating effect of AMD3100 on bone morphogenetic protein-2 induced bone regeneration.

BACKGROUND: AMD3100, a CXCR4 antagonist, is currently prescribed for activating the mobilization of hematopoietic stem cells. Recently, AMD3100 was shown to potentiate bone morphogenetic protein-2 (BMP-2)-induced bone formation by stimulating the trafficking of mesenchymal cells. However, optimization of the strategic combination of AMD3100 and BMP-2 has not yet been clearly established. The purpose of this study was to evaluate the effect of AMD3100 on BMP-2-induced bone regeneration in vitro and in a mouse calvarial defect healing model. METHODS: In vitro osteoblastic differentiation and cell migration after sequential treatments with AMD3100 and BMP-2 were analyzed by alkaline phosphatase (ALP) activity, ALP staining, and calcium accumulation. Migration capacity was evaluated after treating mesenchymal cells with AMD3100 and/or BMP-2. A critical-size calvarial defect model was used to evaluate bone formation after sequential or continuous treatment with AMD3100 and BMP-2. The degree of bone formation in the defect was analyzed using micro-computed tomography (micro-CT) and histological staining. RESULTS: Compared with single treatment using either AMD3100 or BMP-2 alone, sequential treatment with AMD3100 followed by BMP-2 on mesenchymal cells increased osteogenic differentiation. Application of AMD3100 and subsequent BMP-2 significantly activated cell migration on mesenchymal cell than BMP-2 alone or AMD3100 alone. Micro-CT and histomorphometric analysis showed that continuous intraperitoneal (IP) injection of AMD3100 resulted significantly increased new bone formation in BMP-2 loaded scaffold in calvarial defect than control groups without AMD3100 IP injection. Additionally, both single IP injection of AMD3100 and subsequent BMP-2 injection to the scaffold in calvarial defect showed pronounced new bone formation compared to continuous BMP-2 treatment without AMD3100 treatment. CONCLUSION: Our data suggest that single or continuous injection of AMD3100 can potentiate BMP-2-induced osteoblastic differentiation and bone regeneration. This strategic combination of AMD3100 and BMP-2 may be a promising therapy for bone regeneration.

Development of an experimental model for radiation-induced inhibition of cranial bone regeneration.

BACKGROUND: Radiation therapy is widely employed in the treatment of head and neck cancer. Adverse effects of therapeutic irradiation include delayed bone healing after dental extraction or impaired bone regeneration at the irradiated bony defect. Development of a reliable experimental model may be beneficial to study tissue regeneration in the irradiated field. The current study aimed to develop a relevant animal model of post-radiation cranial bone defect. METHODS: A lead shielding block was designed for selective external irradiation of the mouse calvaria. Critical-size calvarial defect was created 2 weeks after the irradiation. The defect was filled with a collagen scaffold, with or without incorporation of bone morphogenetic protein 2 (BMP-2) (1 µg/ml). The non-irradiated mice treated with or without BMP-2-included scaffold served as control. Four weeks after the surgery, the specimens were harvested and the degree of bone formation was evaluated by histological and radiographical examinations. RESULTS: BMP-2-treated scaffold yielded significant bone regeneration in the mice calvarial defects. However, a single fraction of external irradiation was observed to eliminate the bone regeneration capacity of the BMP-2-incorporated scaffold without influencing the survival of the animals. CONCLUSION: The current study established an efficient model for post-radiation cranial bone regeneration and can be applied for evaluating the robust bone formation system using various chemokines or agents in unfavorable, demanding radiation-related bone defect models.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum formation on the exposed tooth-root surface is a critical process in periodontal regeneration. Although various therapeutic approaches have been developed, regeneration of integrated and functional periodontal complexes is still wanting. Here, we found that the OCCM30 cementoblasts cultured on fibrin matrix express substantial levels of matrix proteinases, leading to the degradation of fibrin and the apoptosis of OCCM30 cells, which was reversed upon treatment with a proteinase inhibitor, ε-aminocaproic acid (ACA). Based on these findings, ACA-releasing chitosan particles (ACP) were fabricated and ACP-incorporated fibrin (fibrin-ACP) promoted the differentiation of cementoblasts in vitro, as confirmed by bio-mineralization and expressions of molecules associated with mineralization. In a periodontal defect model of beagle dogs, fibrin-ACP resulted in substantial cementum formation on the exposed root dentin in vivo, compared to fibrin-only and enamel matrix derivative (EMD) which is used clinically for periodontal regeneration. Remarkably, the fibrin-ACP developed structural integrations of the cementum-periodontal ligament-bone complex by the Sharpey's fiber insertion. In addition, fibrin-ACP promoted alveolar bone regeneration through increased bone volume of tooth roof-of-furcation defects and root coverage. Therefore, fibrin-ACP can promote cementogenesis and osteogenesis by controlling biodegradability of fibrin, implicating the feasibility of its therapeutic use to improve periodontal regeneration. STATEMENT OF SIGNIFICANCE: Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Sequential Treatment with SDF-1 and BMP-2 Potentiates Bone Formation in Calvarial Defects.

Stromal cell-derived factor-1 (SDF-1) protein and its receptor, CXCR-4, play an important role in tissue repair and regeneration in various organs, including the bone. SDF-1 is indispensable for bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. However, SDF-1 is not needed after the osteogenic induction has been activated. Since the precise condition for the additive effects of combined DF-1 and BMP-2 in bone healing had not been fully investigated, we aimed to determine the optimal conditions for SDF-1- and BMP-2-mediated bone regeneration. We examined the in vitro osteoblastic differentiation and cell migration after sequential treatments with SDF-1 and BMP-2. Based on the in vitro additive effects of SDF-1 and BMP-2, the critical size defects of mice calvaria were treated with these cytokines in various sequences. Phosphate buffered saline (PBS)-, SDF-1-, or BMP-2-soaked collagen scaffolds were implanted into the calvarial defects (n=36). Periodic percutaneous injections of PBS or the cytokine SDF-1 and BMP-2 into the implanted scaffolds were performed on days 3 and 6, postoperatively. Six experimental groups were used according to the types and sequences of the cytokine treatments. After 28 days, the mice were euthanized and bone formation was evaluated with microcomputed tomography and histology. The molecular mechanism of the additive effect of SDF-1 and BMP-2 was evaluated by analyzing intracellular signal transduction through Smad and Erk phosphorylation. The in vitro experiments revealed that, among all the treatments, the treatment with BMP-2 after SDF-1 showed the strongest osteoblastic differentiation and enhanced cell migration. Similarly, in the animal model, the treatment with SDF-1 followed by BMP-2 treatment showed the highest degree of new bone regeneration than any other groups, including the one with continuous BMP-2 treatment. This new bone formation can be partially explained by the activation of Smad and Erk pathways and enhanced cell migration. These results suggest that sequential treatment with the cytokines, SDF-1 and BMP-2, may be a promising strategy for accelerating bone regeneration in critical size defects.

Effect of dual treatment with SDF-1 and BMP-2 on ectopic and orthotopic bone formation.

PURPOSES: The potent stem cell homing factor stromal cell-derived factor-1 (SDF-1) actively recruits mesenchymal stem cells from circulation and from local bone marrow. It is well established that bone morphogenetic protein-2 (BMP-2) induces ectopic and orthotopic bone formation. However, the exact synergistic effects of BMP-2 and SDF-1 in ectopic and orthotopic bone regeneration models have not been fully investigated. The purpose of this study was to evaluate the potential effects of simultaneous SDF-1 and BMP-2 treatment on bone formation. MATERIALS AND METHODS: Various doses of SDF-1 were loaded onto collagen sponges with or without BMP-2.These sponges were implanted into subcutaneous pockets and critical-size calvarial defects in C57BL/6 mice. The specimens were harvested 4 weeks post-surgery and the degree of bone formation in specimens was evaluated by histomorphometric and radiographic density analyses. Osteogenic potential and migration capacity of mesenchymal cells and capillary tube formation of endothelial cells following dual treatment with SDF-1 and BMP-2 were evaluated with in vitro assays. RESULTS: SDF-1-only-treated implants did not yield significant in vivo bone formation and SDF-1 treatment did not enhance BMP-2-induced ectopic and orthotopic bone regeneration. In vitro experiments showed that concomitant use of BMP-2 and SDF-1 had no additive effect on osteoblastic differentiation, cell migration or angiogenesis compared to BMP-2 or SDF-1 treatment alone. CONCLUSIONS: These findings imply that sequence-controlled application of SDF-1 and BMP-2 must be further investigated for the enhancement of robust osteogenesis in bone defects.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration.

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Analysis of histone deacetylase inhibitor-induced responses in human periodontal ligament fibroblasts.

Periodontal ligament (PDL) fibroblasts play critical roles in the regeneration of periodontal tissues damaged by periodontitis. Histone deacetylase inhibitors (HDIs) have been suggested to be potential tools in tissue engineering. The feasibility of using the HDI, sodium butyrate (NaB) for periodontal regeneration was examined by evaluating its effect on the osteogenic differentiation of human PDL fibroblasts and its modulation of the inflammatory responses to lipopolysaccharide (LPS). NaB did not cause significant cell death at 100 µM but promoted the expression of the osteoblast phenotype (Runx2, osterix, osteocalcin, and bone sialoprotein). NaB significantly inhibited the LPS-induced production of reactive oxygen species and the expression of pro-inflammatory cytokines (IL-1ß and TNF-α). These results suggest that HDIs can offer a potential therapeutic agent for periodontal regeneration.

Insulin-like growth factor 2 promotes osteogenic cell differentiation in the parthenogenetic murine embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent and can differentiate into all somatic cell types. ESCs are an alternative solution to hard tissue regeneration and skeletal tissue repair to treat bone diseases and defects using regenerative strategies. Parthenogenetic ESCs (PESCs) may be a useful alternative stem cell source for tissue repair and regeneration. The defects in full-term development of this cell type enable researchers to avoid the ethical concerns related to ESC research. Moreover, in female patients, if the PESCs are derived from oocytes, then they will have that patient's genetic information. Here, we present data demonstrating that osteogenic differentiation of PESCs can be promoted by insulin-like growth factor 2 (IGF2). PESCs were plated onto Petri dishes with ESC culture medium supplemented with or without IGF2, followed by culturing of the cells for 1 week. PESCs formed floating aggregates called embryoid bodies (EBs). An osteogenic lineage was induced from the EBs by incubating them in medium containing serum, ascorbic acid, ß-glycerophosphate, and retionic acid, with or without IGF2, for 20 days. Gene expression of specific osteoblastic markers such as osteocalcin, osteopontin, osteonectin, bone sialoprotein, collagen type-I, alkaline phosphatase, and Runx2 (Cbfa-I) was analyzed by real-time polymerase chain reaction. The expression level of osteocalcin, osteopontin, osteonectin, and alkaline phosphatase was twofold higher in IGF2-treated PESC derivatives than IGF2-naive PESC derivatives. In vivo experiments were also performed using a critical-sized calvarial defect mouse model. Ten weeks after cell transplantation, more bone tissue regeneration was observed in the IGF2-treated PESC transplantation group than in IGF2-naive PESC transplantation group. Both our in vitro and in vivo data indicate that IGF2 induces osteogenic differentiation of PESCs. Addition of IGF2 may reactivate imprinting genes in PESCs that are only expressed in the paternal genome and are normally silent in PESCs. Our findings provide insights into the mechanisms of skeletal tissue repair and the imprinting mechanisms active in stem cells.

Electrospun silk fibroin scaffolds with macropores for bone regeneration: an in vitro and in vivo study.

We developed three-dimensional electrospun silk fibroin (ESF) scaffolds with controllable pore size. The purpose of this study was to evaluate ESF scaffolds with pores (P-ESF) for bone regeneration via in vitro and in vivo studies, with a comparison to a commercially available porous three-dimensional polylactic acid (PLA) scaffold. P-ESF supported significantly higher proliferation and alkaline phosphatase activity of osteoblasts than PLA in vitro (p < 0.05). Moreover, higher expression levels of activated adhesion-related proteins, including focal adhesion kinase, were observed in the P-ESF than in PLA, as confirmed by western blot analyses. Microcomputed tomography revealed that 78.30% of the original bone volume was attained in the P-ESF implantation group at 7 weeks after critical bone defect formation in rat calvaria. Comparatively, the PLA implantation group showed only 49.31%. Histological evaluation also showed new bone tissue formation upon P-ESF implantation. Taken together, the P-ESF scaffold may be a good bone substitute for bone regeneration.

Modulation of the resorption and osteoconductivity of alpha-calcium sulfate by histone deacetylase inhibitors.

Calcium sulfate (CS) is an osteoconductive material with a long history of clinical use. However, its resorptive properties are not optimal for bone regeneration. Recently, histone deacetylase inhibitors (HDIs) have been suggested as bone regeneration tools. In this study, we investigated the effects of the HDIs sodium butyrate and trichostatin A on alpha-form CS (alphaCS) performance. MC3T3-E1 pre-osteoblasts cultured on alphaCS containing either HDI (alphaCS/HDI) showed higher levels of alkaline phosphatase activity than those cultured on alphaCS alone. The expression of genes characteristic of the osteoblast phenotype, including Runx2, osteocalcin, and bone sialoprotein, was strongly promoted by alphaCS/HDI. When cultured on alphaCS/HDIs, the osteoclastic differentiation of RAW264.7 monocytes was substantially suppressed, as measured by tartrate-resistant acid phosphatase (TRAP) activity and the expression levels of calcitonin receptor and TRAP. Neither HDI affected the CS setting time, compressive strength, or dissolution in a simulated body fluid. In a rat calvarial model of critical size bone defects, alphaCS/HDIs enhanced osteoblast differentiation, led to new bone formation, and delayed resorption, as confirmed by micro-computed tomography and histological analyses.

Comparative evaluation of different crystal-structured calcium sulfates as bone-filling materials.

The mechanical and handling properties and biological performances of two types of calcium sulfate (betaCS and alphaCS) as bone-filling materials were compared. The influence of two modifiers such as hydroxypropylmethylcellose (HPMC) and fibrin was also examined. alphaCS showed higher strength than, and similar setting time and injectability to those of betaCS. The degradation of CS in a simulated body fluid (SBF) was checked by measuring the amount of calcium released to SBF. alphaCS showed reduced calcium release than betaCS. The modifiers tended to increase the calcium release. The MC3T3-E1 preosteoblasts cultured on alphaCS showed higher levels of alkaline phosphatase (ALP) activity than those cultured on betaCS. alphaCS strongly promoted gene expression of osteoblast phenotypes including Runx2, alpha1(I) collagen, osteocalcin, and bone sialoprotein. There was no significant difference in cell adhesion and proliferation between two types of CS. The addition of modifiers to CS increased cell proliferation, ALP activity, and the gene expression. The osteoclastic differentiation of RAW264.7 monocytes was checked. The cells on both types of CS produced tartrate-resistant acid phosphatase (TRAP) activity with no significant difference. These cell response results indicated that alphaCS promoted osteoblast differentiation over betaCS but not osteoclast differentiation. Conclusively, a particular form of commercially available alphaCS possesses superior properties to betaCS in terms of mechanical properties and supports for osteoblast differentiation, suggesting that alphaCS could be an alternative to the conventionally used betaCS. The addition of HPMC and fibrin could further improve the feasibility of alphaCS as a bone-filling material.

Comparative evaluation of nanofibrous scaffolding for bone regeneration in critical-size calvarial defects.

In a previous study we found that nanofibrous poly(l-lactic acid) (PLLA) scaffolds mimicking collagen fibers in size were superior to solid-walled scaffolds in promoting osteoblast differentiation and bone formation in vitro. In this study we used an in vivo model to confirm the biological properties of nanofibrous PLLA scaffolds and to evaluate how effectively they support bone regeneration against solid-walled scaffolds. The scaffolds were implanted in critical-size defects made on rat calvarial bones. Compared with solid-walled scaffolds, nanofibrous scaffolds supported substantially more new bone tissue formation, which was confirmed by micro-computed tomography measurement and von Kossa staining. Goldner's trichrome staining showed abundant collagen deposition in nanofibrous scaffolds but not in the control solid-walled scaffolds. The cells in these scaffolds were immuno-stained strongly for Runx2 and bone sialoprotein (BSP). In contrast, solid-walled scaffolds implanted in the defects were stained weakly with trichrome, Runx2, and BSP. These in vivo results demonstrate that nanofibrous architecture enhances osteoblast differentiation and bone formation.

The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling.

The 4-1BB is a costimulatory molecule similar to the receptor activator of NF-kappaB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4-1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4-1BB and 4-1BB ligand (4-1BBL) in osteoclastogenesis was investigated using 4-1BB(-/-) and 4-1BB(+/+) mice. Osteoclast precursors normally express 4-1BB and 4-1BBL after exposure to RANKL, which was confirmed by semi-quantitative RT-PCR and flow cytometry. The 4-1BB(-/- )mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4-1BB(-/-) bone marrow-derived macrophages (BMM) ex vivo. In addition, 4-1BB(-/-) BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c-Fos in response to RANKL stimulation. Retroviral transduction of wild-type as well as partial-length 4-1BB, which lacks TNF receptor-associated factor 2-binding sites for signaling, restored the osteoclastogenic ability of 4-1BB(-/-) BMM. Furthermore, both recombinant 4-1BB and 4-1BBL enhanced RANKL-induced osteoclastogenesis by 4-1BB(+/+) BMM and the induction of c-Fos and NFATc1.Together, these results indicate that 4-1BBL and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling, findings that may delineate the complex nature of the 4-1BBL and 4-1BB interaction.

Development of 3-D nanofibrous fibroin scaffold with high porosity by electrospinning: implications for bone regeneration.

We made a three-dimensional (3-D) nanofibrous fibroin scaffold (NFS) with high porosity (94%) and examined its feasibility in bone regeneration. Under scanning electron microscopy, MC3T3-E1 preosteoblasts on the scaffold showed more spread on the first day after seeding compared with a 2-D scaffold. MTT assay showed significantly increased proliferation in 3-D NFS compared with 2-D NFS 7 days after seeding (P < 0.05). Western immunoblotting for activated paxillin, FAK, AKT, C-Src, and ERK1/2 antibodies showed signals from the extracellular matrix were significantly increased in 3-D NFS. Newly developed 3-D electrospun NFS may be a good candidate for use in bone regeneration.