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Transforming growth factor beta 1 (TGF-β1) is a key morphogen in regenerative endodontics; yet, its location within the hard tissue phase of dentin and its availability in mature roots have not been fully elucidated. Young mature (n = 8) and immature (n = 11) roots from sound premolars were obtained from 13 orthodontic patients aged 17 ± 1 and 12 ± 1 years, respectively. Roots were cleaned of organic remnants in 5% sodium hypochlorite. The width of the minor foramen was measured using a digital microscope. TGF-β1 distribution was assessed in 3 roots per group by immunostaining combined with confocal laser scanning microscopy. The root dentin of the remaining 13 roots was powdered and decalcified in 17% EDTA to determine the overall levels of hard tissue–embedded TGF-β1 by enzyme-linked immunosorbent assay. Data were compared between groups using the Student t test (α =.05). The minor foramen was 168 ± 49 μm versus 557 ± 295 μm in mature compared with immature roots (P <.05). TGF-β1 was highly stainable toward the pulp space in both groups. It was clearly associated with peritubular dentin and apparently absent in nontubular outer dentin. TGF-β1 content was 115 ± 31 pg and 74 ± 35 pg/100 mg mature versus immature root dentin, respectively (P >.05). TGF-β1 is deposited into the peritubular dentin. It should be possible to release this molecule in regenerative endodontic procedures from young mature roots as well as immature roots. [ABSTRACT FROM AUTHOR]

Impaired bone regeneration by excess inflammation leads to failure of bone healing. Current therapies display limited benefits making new treatments imperative. Our recent discoveries of the anti-inflammatory characteristics of bromodomain and extra terminal domain (BET) inhibitors, N -methylpyrrolidone (NMP) and N , N -Dimethylacetamide (DMA), implicate possible therapeutic use of epigenetic drugs in inflammation-impaired bone healing. Here, we investigated the effects of NMP and DMA on osteogenesis in vitro and ex vivo under the influence of TNFα, a key cytokine responsible for impaired fracture healing. NMP and DMA pre-treatment recovered TNFα-inhibited expression of essential osteoblastic genes, Alp, Runx2, and Osterix as well as mineralization in multipotent stem cells, but not in pre-osteoblasts and calvarial osteoblasts. The mechanism of action involves the recovery of TNFα-suppressed BMP-induced Smad signaling and the reduction of TNFα-triggered ERK pathway. In addition, ERK inhibitor treatment diminished the effect of TNFα on osteogenesis, which reinforces the role of ERK pathway in the adverse effect of TNFα. Furthermore, endochondral ossification was analyzed in an ex vivo bone culture model. TNFα largely abrogated BMP-promoted growth of mineralized bone while pre-treatment of NMP and DMA prevented the deleterious effect of TNFα. Taken together, these data shed light on developing low- affinity epigenetic drugs as new therapies for inflammation-compromised bone healing. • NMP and DMA protect MSCs and osteoprogenitors from the inhibitory effect of TNFα on osteogenesis. • TNFα inhibits osteogenesis by an ERK-dependent mechanism. • Mechanism of action of NMP and DMA is Smad- and ERK-dependent, but NF-κB-independent. • Low-affinity epigenetic inhibitors are promising candidates for new treatments for inflammation-impaired bone healing. [ABSTRACT FROM AUTHOR]

Abstract Introduction In carious teeth, transforming growth factor beta 1 (TGF-β1) is released from the dentin matrix and possibly activated in an acidic environment. Conversely, EDTA solutions with a neutral to slightly alkaline pH are used in clinics to promote cell homing in regenerative endodontic procedures. We hypothesized that citric acid (CA) might be more beneficial. Methods TGF-β1 release from human dentin disks conditioned with either 10% CA (pH = 2) or 17% EDTA (pH = 8) and the behavior of human stem cells toward such pretreated dentin were studied. The protein concentration in conditioning solutions after 10 minutes of dentin exposure was determined using a pH-independent slot blot technique. Results There was a 5-fold higher concentration of the target protein in CA (382 ± 30 ng/disk) compared with EDTA (66 ± 3 ng/disk, P <.005). Using confocal laser scanning microscopy on immunofluorescent-labeled disks, we identified a high density of TGF-β1 in peritubular dentin after CA treatment. A migration assay showed that CA conditioning attracted significantly more stem cells toward the dentin after 24 hours compared with EDTA (P <.05) or phosphate-buffered saline (P <.005). To investigate whether the cell response to these dentin surfaces could be affected by different pretreatments, we cultured stem cells on conditioned dentin disks and found that CA had a significantly (P <.05) better effect than EDTA on cell attachment and cell survival. Conclusions CA conditioning could be useful and may have significant benefits over current treatments. Highlights • Under the conditions of the current study using human dentin disks, a 10% citric acid solution (pH = 2) was significantly (P <.05) better than 17% EDTA (pH = 8) regarding the following: -The release of transforming growth factor beta 1 (TGF-β1) into solution. -The exposure of TGF-β1 on the dentin surface. -The attraction of stem cells toward conditioned dentin. -Stem cell attachment on the conditioned surface. -Stem cell survival. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: In pulpless immature human premolars implanted in rodents, this study investigated whether fibrin gel offered advantages over leaving the root canal empty regarding soft tissue ingrowth and cell differentiation. Methods: Root canals of extracted human immature premolars (n = 12) were accessed and then irrigated with 5% sodium hypochlorite followed by 17% ethylenediaminetetraacetic acid. Root canals were then either left empty or filled with a fibrin gel (n = 6 each) before being placed subcutaneously on top of the calvarial bone of rats (1 tooth per rat) for 12 weeks. After sacrifice, teeth were histologically assessed. Tissue ingrowth was quantified and compared between groups using the Mann-Whitney U test (P < .05). Cells adhering to the pulp canal wall were immunohistochemically screened for the presence of bone sialoprotein (BSP) and dentin sialoprotein (DSP). Results: More tissue grew into the pulp space when teeth were filled with fibrin gel (P < .05). The presence of fibrin gel affected not only the extent of tissue ingrowth but also tissue morphology and differentiation of cells contacting the dentinal wall. In the fibrin gel group, newly formed tissue was similar to normal pulp, constituted of inner pulp, cell-rich zone, cell-free zone, and an apparent odontoblast layer, which stained positive for BSP and DSP. Newly formed blood vessels were also more abundant compared with the initially empty root canals. Conclusions: Under the conditions of this study, fibrin gel improved cell infiltration and cell-dentin interaction. Both are necessary for pulp tissue regeneration. [Copyright &y& Elsevier]

Abstract: Bone morphogenetic proteins (BMP), in particular BMP-2, are the growth factors primarily responsible for osteoinduction. A knowledge of interactions between bone substitute materials and growth factor variants is crucial to designing bone substitutes with an ideal release profile. Here we compare glycosylated and non-glycosylated recombinant human bone morphogenetic protein-2 (rhBMP-2) either incorporated into a hydrolyzable polyethylene glycol (PEG) hydrogel developed as a slow release system or adsorbed to a deproteinized bovine bone matrix (DBBM), a clinically well-established bone substitute material. rhBMP-2 loaded materials were immersed in cell culture medium and rhBMP-2 concentration profiles in the supernatant were determined by an enzyme-linked immunosorbent assay. The corresponding biological activities were assessed in vitro by alkaline phosphatase activity assay. We show a strong affinity of rhBMP-2 for DBBM and reduced biological activity after its release from PEG hydrogels. Glycosylated rhBMP-2 was significantly less affected by the hydrogel and interacted significantly more strongly with DBBM than non-glycosylated rhBMP-2. We therefore question the combination of PEG hydrogels with DBBM as a rhBMP-2 delivery system over DBBM alone, since rhBMP-2 released from the hydrogel will be trapped by DBBM. Moreover, our results suggest that glycosylated rhBMP-2 is favorable in combination with PEG hydrogels, since its activity is better preserved, whereas in combination with DBBM non-glycosylated rhBMP-2 is favorable, benefiting from an initially higher concentration of free rhBMP-2. [Copyright &y& Elsevier]

Synthetic bone substitutes are used for regeneration of bone defects and for vertical or horizontal bone augmentation. With the development of new manufacturing methodologies such as additive manufacturing, not only can the outer shape of a scaffold (the macroarchitecture) be personalized, but also the microarchitecture, defined as the distribution of material within the scaffold, can be designed and optimized for bone ingrowth. Our study assessed the optimal pore-based microarchitecture for osteoconduction in comparison to vertical bone augmentation. Histological analysis of the samples from an osteoconduction and a bone augmentation model showed that the best microarchitecture to achieve vertical bone augmentation contained pores of 1.7 mm diameter and differed from the optimal microarchitecture for osteoconduction, which contained pores of 1.2 mm diameter. The extent of vertical bone augmentation correlated with increasing pore diameter. Moreover, the differences between the microarchitectures were highly significant for osteoconduction, but less significant for vertical bone augmentation. Thus, our results suggest that the microarchitectures of the scaffolds in vertical bone augmentation and in bone regeneration of a defect are crucial determinants of the depth of bone ingrowth and the area of bony regeneration. However, the microarchitecture appears to be more crucial in osteoconduction. [Display omitted] • Optimal microarchitectures enhance bone augmentation outcomes. • The microarchitecture is a more crucial determinant in osteoconduction than in bone augmentation. [ABSTRACT FROM AUTHOR]

Histomorphometry and microCT are the two dominant imaging techniques to study bone structure and quality to evaluate repair, regeneration, and disease. These two methods are complementary; where histology provides highly resolved tissue properties on a cellular level in 2D, microCT provides spatial information of bone micro-structure in 3D. For this reason, both of these modalities are commonly used in bone studies. As it is not trivial to combine the images of these two modalities, the two methods are typically applied to different specimens within a study. However, we believe that applying both imaging modalities to the same specimen with a suitable fusion strategy may further strengthen the value of each modality. Therefore, we propose a registration method to align 2D histology slices with a 3D microCT volume, without any prior knowledge of the sectioning direction. In a preprocessing step, bone is extracted from both images. Then, we use a strategy for initializing potential locations, and an iterative approach for searching for an ideal fitting plane using Radon-based rigid transforms and feature-based affine alignments. The algorithm was tested and validated with simulated and real data. For the latter, microCT images of trabecular bone with 76 corresponding histological sections acquired from decalcified and calcified specimens were used. The registration resulted in 94.7% acceptable solutions as defined by a registration orientation error of less than 3°. Average registration accuracy of the acceptable results was 0.6°, leading to a target registration error for our method of 106.3 μm, computed based on landmarks annotated by an observer. This corresponds roughly to 10 pixels in the images; although, the relation to actual visible structures that provide the features to register, is arguably more relevant. [ABSTRACT FROM AUTHOR]

Healing large bone defects remains challenging in orthopedic surgery and is often associated with poor outcomes and complications. A major issue with bioengineered constructs is achieving a continuous interface between host bone and graft to enhance biological processes and mechanical stability. In this study, we have developed a new bioengineering strategy to produce oriented biocompatible 3D PLGA/aCaP nanocomposites with enhanced osseointegration. Decellularized scaffolds -containing only extracellular matrix- or scaffolds seeded with adipose-derived mesenchymal stromal cells were tested in a mouse model for critical size bone defects. In parallel to micro-CT analysis, SAXS tensor tomography and 2D scanning SAXS were employed to determine the 3D arrangement and nanostructure within the critical-sized bone. Both newly developed scaffold types, seeded with cells or decellularized, showed high osseointegration, higher bone quality, increased alignment of collagen fibers and optimal alignment and size of hydroxyapatite minerals. [ABSTRACT FROM AUTHOR]

Abstract: Objective: This study investigated effects of cerium-chloride on fibroblast and osteoblast differentiation and proliferation. Methods: MC3T3-E1 cells were plated for an alkaline phosphatase (ALP) activity test. On day 3, CeCl3-solutions (1, 5 or 10, w/v%) were added. After 10s, the solutions were aspirated and washed to remove residual CeCl3. On day 6 ALP activity was determined. Cell activity and proliferation was assessed by thiazolyl blue tetrazolium dye reduction assay (MTT-test) also 3 days after exposure to the CeCl3-solutions. Calcium deposition by preosteoblastic cells was determined 4 weeks after the exposure of the cells by alizarin red staining. Furthermore, in all experiments the influence of adding rhBMP-2 was tested. Statistical analysis was performed by repeated-measures ANOVA using the post hoc Fisher least significant difference (LSD) test. Statistical significance was set at p <0.05. Results: Exposure to a Ce-solution of 1% or higher reduced ALP activity significantly. The addition of rhBMP-2 was able to elevate ALP activity above control level. MTT-test showed a significant decrease in cellular activity by 5% Ce or higher. The addition of rhBMP-2 had no positive effect. For human foreskin fibroblasts, exposure to even 10% Ce yielded a significant increase in cellular activity. Ce reduced calcium deposition to a level of below 50% of the control. The addition of rhBMP-2 restored mineral deposition to control levels for all Ce concentrations. Conclusion: CeCl3 had a stimulating effect on fibroblasts but a depressing influence on osteoblasts. However, adding rhBMP-2 could compensate the latter influence. [Copyright &y& Elsevier]

Abstract: Articular cartilage undergoes complex loading modalities generally including sliding, rolling and plowing (i.e. the compression by a condyle normally to the tissue surface under simultaneously tangential displacement, thus generating a tractional force due to tissue deformation). Although in in vivo studies it was shown that excessive plowing can lead to osteoarthritis, little quantitative experimental work on this loading modality and its mechanobiological effects is available in the literature. Therefore, a rolling/plowing explant test system has been developed to study the effect on pristine cartilage of plowing at different perpendicular forces. Cartilage strips harvested from bovine nasal septa of 12-months-old calves were subjected for 2h to a plowing-regime with indenter normal force of 50 or 100N and a sliding speed of 10mms−1. 50N produced a tractional force of 1.2±0.3N, whereas 100N generated a tractional force of 8.0±1.4N. Furthermore, quantitative-real-time polymerase chain reaction experiments showed that TIMP-1 was 2.5x up-regulated after 50N plowing and 2x after 100N plowing, indicating an ongoing remodeling process. The expression of collagen type-I was not affected after 50N plowing but it was up-regulated (6.6x) after 100N plowing, suggesting a possible progression to an injury stage of the cartilage, as previously reported in cartilage of osteoarthritic patients. We conclude that plowing as performed by our mimetic system at the chosen experimental parameters induces changes in gene expression depending on the tractional force, which, in turn, relates to the applied normal force. [Copyright &y& Elsevier]

Regulation of RANKL (receptor activator of nuclear factor ?B ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption. [ABSTRACT FROM AUTHOR]

Abstract: Osteoarthritis is a chronic degenerative disease affecting body joints. Abnormal mechanical loading could be an initiating factor of cartilage damage, by influencing chondrocytes activity. To date, devices performing mechanical studies of viable tissues are mostly uniaxial. In this work, we developed and validated a multi-axial device for static and dynamic mechanical testing of viable soft tissues. The system, named RPETS, is composed of a motor driven indenter, moving vertically and horizontally along the bottom of a tank containing tissue samples and it can apply combined compression, sliding, and rolling on viable samples. Validation studies were performed with standard rubber and bovine nasal cartilage tissue. Static tests demonstrated that the system is comparable to existing uniaxial devices, with a maximum force control error smaller than 0.5N and a positioning resolution of 5μm. Dynamic tests performed with different motion profiles showed that the system can exert a load of 100N with a maximum velocity of 100mm/s maintaining the force control error within 10% of the desired value. Sinusoidal motion frequency can vary between 0.05 and 0.5Hz. In practical tests, viability staining of dynamically loaded cartilage slices showed extents of cell death to depend on the indenter velocity. [Copyright &y& Elsevier]

Abstract: Static stretching is a major type of mechanical stimuli utilized during distraction osteogenesis (DO), a general surgical method for the lengthening of bone. The molecular signals that drive the regenerative process in DO include a variety of cytokines. Among these, bone morphogenic protein (BMP, -2 and -4) has been reported to exhibit strongly enhanced expression following the application of mechanical strain during the distraction phase. We hypothesize that mechanical stretching enhances osteoblast differentiation in DO by means of interaction with BMP-2 induced cytokine stimulation. C2C12 pluripotential myoblasts were exposed to stretching load and the resulting cell proliferation and osteoblast differentiation were then examined. The application of static stretching force resulted in significant cell proliferation at day 3, although with variable intensity according to the magnitude of stretching. A combined treatment of stretching load with BMP-2 stimulation significantly increased alkaline phosphatase (ALP) activity and up-regulated the gene expression of osteogenic markers (ALP, type I collagen, osteopontin, osteocalcin, cbfa1, osterix and dlx5). Results obtained with the combined treatment yielded more activity than just the BMP-2 treatment or stretching alone. These results reveal that specific levels of static stretching force increase cell proliferation and effectively stimulate the osteoblast differentiation of C2C12 cells in conjunction with BMP-2 stimulation, thus indicating a synergistic interaction between mechanical strain and cytokine signaling. [Copyright &y& Elsevier]

Abstract: The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering. [Copyright &y& Elsevier]

N, N-Dimethylacetamide is an FDA approved solvent widely used in pharmaceutical industry to facilitate the solubility of lipophilic, high molecular weight drugs with poor water solubility. However, the cytotoxic effects of DMA raises the concern about its use in clinical applications. In the present study, we address the effect of DMA on spermatogenesis. Male Sprague Dawley rats were injected intra-peritoneally for 8 weeks, once a week at a dose of 862 mg/kg. Analysis of reproductive parameters revealed that DMA treated animals exhibit spermatid formation defects within the testis describing the characteristics of oligozoospermia. A subsequent decrease in epididymal sperm concentration along with distortion of sperm morphology was observed. The mitochondrial and microtubule organization in the sperm is considerably modified by DMA. This disrupts the sperm kinetics thus decreasing the total and progressive sperm motility. Finally, DMA treatment resulted in loss of fertility. Our results indicate that exposure to DMA has a negative impact on spermatogenesis and leads to infertility in male rats by inhibiting the post meiotic stages of sperm development. Therefore, the use of DMA in humans must be closely monitored. • N, N-Dimethylacetamide (DMA) alters the ultrastructure of acrosome leading to infertility in rats. • DMA impairs spermatogenesis by inhibiting the post-meiotic round and elongated spermatids within the testis. • DMA distorts the mitochondrial and microtubular structure of sperm therefore, reducing the sperm motility. [ABSTRACT FROM AUTHOR]