BMP-2 Induced Signaling Pathways and Phenotypes: Comparisons Between Senescent and Non-senescent Bone Marrow Mesenchymal Stem Cells.

The use of BMP-2 in orthopedic surgery is limited by uncertainty surrounding its effects on the differentiation of mesenchymal stem cells (MSCs) and how this is affected by cellular aging. This study compared the effects of recombinant human BMP-2 (rhBMP-2) on osteogenic and adipogenic differentiation between senescent and non-senescent MSCs. Senescent and non-senescent MSCs were cultured in osteogenic and adipogenic differentiation medium containing various concentrations of rhBMP-2. The phenotypes of these cells were compared by performing a calcium assay, adipogenesis assay, staining, real-time PCR, western blotting, and microarray analysis. rhBMP-2 induced osteogenic differentiation to a lesser extent (P < 0.001 and P = 0.005 for alkaline phosphatase activity and Ca2+ release) in senescent MSCs regardless of dose-dependent increase in both cells. However, the induction of adipogenic differentiation by rhBMP-2 was comparable between them. There was no difference between these two groups of cells in the adipogenesis assay (P = 0.279) and their expression levels of PPARγ were similar. Several genes such as CHRDL1, NOG, SMAD1, SMAD7, and FST encoding transcription factors were proposed to underlie the different responses of senescent and non-senescent MSCs to rhBMP-2 in microarray analyses. Furthermore, inflammatory, adipogenic, or cell death-related signaling pathways such as NF-kB or p38-MAPK pathways were upregulated by BMP-2 in senescent MSCs, whereas bone forming signaling pathways involving BMP, SMAD, and TGF- ß were upregulated in non-senescent MSCs as expected. This phenomenon explains bone forming dominance by non-senescent MSCs and possible frequent complications such as seroma, osteolysis, or neuritis in senescent MSCs during BMP-2 use in orthopedic surgery.

Three-dimensional printed polylactic acid scaffold integrated with BMP-2 laden hydrogel for precise bone regeneration.

BACKGROUND: Critical bone defects remain challenges for clinicians, which cannot heal spontaneously and require medical intervention. Following the development of three-dimensional (3D) printing technology is widely used in bone tissue engineering for its outstanding customizability. The 3D printed scaffolds were usually accompanied with growth factors, such as bone morphometric protein 2 (BMP-2), whose effects have been widely investigated on bone regeneration. We previously fabricated and investigated the effect of a polylactic acid (PLA) cage/Biogel scaffold as a carrier of BMP-2. In this study, we furtherly investigated the effect of another shape of PLA cage/Biogel scaffold as a carrier of BMP-2 in a rat calvaria defect model and an ectopic ossification (EO) model. METHOD: The PLA scaffold was printed with a basic commercial 3D printer, and the PLA scaffold was combined with gelatin and alginate-based Biogel and BMP-2 to induce bone regeneration. The experimental groups were divided into PLA scaffold, PLA scaffold with Biogel, PLA scaffold filled with BMP-2, and PLA scaffold with Biogel and BMP-2 and were tested both in vitro and in vivo. One-way ANOVA with Bonferroni post-hoc analysis was used to determine whether statistically significant difference exists between groups. RESULT: The in vitro results showed the cage/Biogel scaffold released BMP-2 with an initial burst release and followed by a sustained slow-release pattern. The released BMP-2 maintained its osteoinductivity for at least 14 days. The in vivo results showed the cage/Biogel/BMP-2 group had the highest bone regeneration in the rat calvarial defect model and EO model. Especially, the bone regenerated more regularly in the EO model at the implanted sites, which indicated the cage/Biogel had an outstanding ability to control the shape of regenerated bone. CONCLUSION: In conclusion, the 3D printed PLA cage/Biogel scaffold system was proved to be a proper carrier for BMP-2 that induced significant bone regeneration and induced bone formation following the designed shape.

Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3D-Encapsulated Co-Culture Environment.

BACKGROUND: Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix® 3D culture system, which provides a physiologically relevant environment. METHODS: The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. RESULTS: Treatment with osteoblastic induction factors (+3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D co-culture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK-RANKL signaling, which is important in osteoblast regulation of osteoclasts. CONCLUSION: In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

A novel 3D indirect co-culture system based on a collagen hydrogel scaffold for enhancing the osteogenesis of stem cells.

In this study, the paracrine effect between adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts was investigated in collagen-based three-dimensional (3D) scaffolds. 3D encapsulation of mesenchymal stem cells in hydrogel scaffolds was conducted for bone tissue regeneration. Osteoblasts were encapsulated in alginate microbeads with uniform size, which could be controlled by varying the supply voltage using electrostatic droplet extrusion. Osteoblast-encapsulated microbeads were embedded with ADSCs in collagen bulk hydrogel scaffolds with a high survival rate. The separated space between the two types of cells made it possible to confirm ADSC differentiation into osteogenic lineages in the 3D collagen hydrogel scaffold by the paracrine effect in vitro. Furthermore, co-cultured ADSC and osteoblasts showed enhanced bone formation compared with the ADSC monoculture group in the rat calvarial defect model. The system developed in this study provides a novel in vitro tissue model for bone regeneration without exogenous factors, and it has the potential to be used to study the paracrine effect in various co-culture systems in the near future.

BMP-2 and hMSC dual delivery onto 3D printed PLA-Biogel scaffold for critical-size bone defect regeneration in rabbit tibia.

3D printing technology has various advantages, and the incorporation of bioactive substances into the 3D printed scaffold provides the biological and architectural characteristics of the scaffolds, which is very important for obtaining a good osseointegration effect. In this relation, this study prepared a novel porous hollow cage poly(lactic acid) (PLA) 3D printed scaffold and combined recombinant human bone morphogenetic protein-2 (rhBMP-2) and/or mesenchymal stem cells (MSCs) with Biogel composed of gelatin and alginate. Then, the scaffolds were used to evaluate the resulting bone regeneration through both in vitro and in vivo tests. The experimental group was divided into four groups as follows: only PLA scaffold (PLA); PLA scaffold filled with BMP-2 loaded on Biogel (P-BG-B2); PLA scaffold filled with MSCs encapsulated Biogel (P-BG-M); PLA scaffold filled with both BMP-2 and MSCs loaded on Biogel (P-BG-B2-M). Then in vitro results showed that the PLA-Biogel-based scaffold increased cell proliferation, and the P-BG-B2-M group showed a higher alkaline phosphatase activity and bone-related gene expression than was seen with the P-BG-M group at all the time points. It was shown that four weeks post-operative micro-CT analysis showed that within the defect site the P-BG-B2 group had a significantly higher percent bone volume (BV/TV) than the PLA group and P-BG-M group. And, out of the defect site, the P-BG-B2-M group BV/TV was shown significantly higher than the PLA group (p < 0.05). Histologically, defects in the P-BG-B2-M group showed a homogeneous new bone distribution, however the P-BG-B2 group and P-BG-M group presented a notably higher bone formation in the internal region than in the proximal region of the bone defect site. In conclusion, the 3D PLA-Biogel-based scaffold adapted rhBMP-2 and MSCs with carrier PLA showed good biocompatibility and high possibility as an effective and satisfactory bone graft material.

Enhanced healing of rat calvarial defects with 3D printed calcium-deficient hydroxyapatite/collagen/bone morphogenetic protein 2 scaffolds.

In this paper, we mainly to evaluate the newly formed bone using the Calcium deficient hydroxyapatite (CDHA)/collagen-based bio-ceramic scaffold as Bone Morphogenetic Protein-2 (BMP-2) carrier in rat calvarial critical-sized bone defect. In the real-time PCR analysis, the CDHA/collagen scaffold loaded rhBMP-2 group showed significantly enhanced results of bone-related gene expression (p < 0.05). In the in vivo study, the micro-CT showed that the main bone formation parameters of percent bone volume and trabecular number of the two experiment groups (CDHA/Collagen (CDHA) group, BV/TV: 14.21 ± 3.20, Tb.N: 2.37 ± 0.50; CDHA/Collagen/rhBMP-2(BMP) group, BV/TV: 14.51 ± 3.12, Tb.N: 2.75 ± 0.65) were significantly higher than those of the control (Blank, BV/TV: 3.25 ± 1.25, Tb.N: 0.57 ± 0.20) group (p < 0.05). Although there was no significant difference between the two experimental groups, the BMP group results were slightly higher than those of the CDHA group (p > 0.05). Moreover, the histological results also supported the micro-CT results. The scaffold of CDHA/collagen seems to be a suitable bio-ceramic carrier loaded rhBMP-2, and appears to enhance new bone formation and bone regeneration in bone defect after implantation.

Positive Effects of Bisphosphonates on Osteogenic Differentiation in Patient-Derived Mesenchymal Stem Cells for the Treatment of Osteoporosis.

BACKGROUND: Recent evidence from in vitro and in vivo studies indicates that bisphosphonates may promote osteoblastic bone formation and potently inhibit osteoclast activity. However, little is known about the potential effect of bisphosphonates on the recruitment of osteoblastic precursors from patient-derived bone marrow stromal cells due to difficulties in accessing human bone marrow from healthy and disease subjects. METHODS: In this study, we evaluated the potential of using FDA-approved and clinically utilized bisphosphonates such as alendronate, ibandronate, and zoledronate to enhance the development of bone forming osteoblasts from osteoporosis patient- and healthy-person derived hBMSCs (op-MSCs and hp-MSCs, respectively). hBMSCs were obtained from postmenopausal women without endocrine diseases or receiving hormone replacement therapy. Cells were treated with or without a bisphosphonate (alendronate, ibandronate, and zoledronate) and analyzed over 21 days of culture. RESULTS: hBMSC from osteoporosis-patient with bisphosphonates treatment demonstrated a significant increase in Alizarin red staining after 7 days compared to that from healthy-person. Calcium contents and alkaline phosphatase (ALP) enzyme activity also demonstrated an increased propensity in hMSCs from osteoporosis-patient compared to those from healthy-person, although there were inter-individual variations. Gene expression levels varied among different donors. There were no significant differences in the effect on the osteoblastic differentiation of hBMSCs among alendronate, ibandronate, and zoledronate. Statistical significance in the osteoblastic differentiation of hBMSCs between the positive control group cultured in osteogenic medium alone and groups cultured in osteogenic medium supplemented with bisphosphonate was not shown either. These results might be due to various cell types of hBMSCs from individual clinical patients and concentrations of bisphosphonate used. CONCLUSION: Our study using a clinically relevant in vitro model suggests that bisphosphonate treatment is more effective for patients with osteoporosis than its preventive effect for healthy person. In addition, patient-specific responses to bisphosphonates should be considered rather than bisphosphonate type prior to prescription. Further investigations are needed to determine how bisphosphonates influence hBMSCs function to mediate bone quality and turnover in osteoporotic patients. Such studies can generate novel approaches to treat age-related osteoporotic bone loss.

Cold-spray coating of hydroxyapatite on a three-dimensional polyetheretherketone implant and its biocompatibility evaluated by in vitro and in vivo minipig model.

PEEK is a bioinert material that does not chemically bind to native bone tissue and thus formation of natural bone-like hydroxyapatite (HA) coating layer on PEEK has been an important challenge to improve biocompatibility and to preserve mechanical property of PEEK. Among various coating techniques, cold-spray coating method is suitable to form stable HA coating layer on PEEK while maintaining their chemical properties, because it can be conducted in relatively low-temperature range. Therefore, in this research, we used cold-spray coating method to form a thick layer of HA on the topographically complex PEEK substrates with periodic ridges on the surface and implanted in iliac bone defects of minipigs which is known to be similar with human body system. In addition, PEEK cage for clinical usage was coated with HA and inserted in the lumbar intervertebral disc space of minipig. We observed higher ALP activity, calcium production, and BSP production of human bone marrow mesenchymal stem cells on the HA-coated PEEK implants than the bare PEEK group in in vitro test. In addition, two-dimensional histological analysis and three-dimensional micro CT analysis demonstrated that implantation of complex shape of HA-PEEK hybrid implant in in vivo minipig model resulted sufficient biocompatibility and osseointegration for further clinical applications. Notably, due to the enhanced stability of PEEK cage induced from HA coating layer, osseointegration rate of the small HA blocks loaded inside the PEEK cage was also significantly improved which indicates overall increased fusion rate and adherence of the HA-coated PEEK cage. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 647-657, 2017.

Inhibition of ß-Catenin to Overcome Endocrine Resistance in Tamoxifen-Resistant Breast Cancer Cell Line.

BACKGROUND: The ß-catenin signaling is important in cell growth and differentiation and is frequently dysregulated in various cancers. The most well-known mechanism of endocrine resistance is cross-talk between the estrogen receptor (ER) and other growth factor signaling, such as phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway. In the present study, we investigated whether ß-catenin could be a potential target to overcome endocrine resistance in breast cancer. METHODS: We established tamoxifen-resistant (TamR) cell line via long-term exposure of MCF-7 breast cancer cells to gradually increasing concentrations of tamoxifen. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of ß-catenin was measured using luciferase activity assay. RESULTS: TamR cells showed a mesenchymal phenotype, and exhibited a relatively decreased expression of ER and increased expression of human epidermal growth factor receptor 2 and the epidermal growth factor receptor. We confirmed that the expression and transcriptional activity of ß-catenin were increased in TamR cells compared with control cells. The expression and transcriptional activity of ß-catenin were inhibited by ß-catenin small-molecule inhibitor, ICG-001 or ß-catenin siRNA. The viability of TamR cells, which showed no change after treatment with tamoxifen, was reduced by ICG-001 or ß-catenin siRNA. The combination of ICG-001 and mTOR inhibitor, rapamycin, yielded an additive effect on the inhibition of viability in TamR cells. CONCLUSION: These results suggest that ß-catenin plays a role in tamoxifen-resistant breast cancer, and the inhibition of ß-catenin may be a potential target in tamoxifen-resistant breast cancer.

The inhibitory effect of zoledronate on early-stage osteoinduction by recombinant human bone morphogenetic protein 2 in an osteoporosis model.

This study evaluated the effect of the combined treatment of intravenous zoledronic acid (ZA, 0.08 mg/kg) and rhBMP-2 (5 µg) on osteogenesis in a calvarial defect model of ovariectomized SD rats. New bone formation was evaluated 4 or 8 weeks after calvarial defect implantation using micro-CT and histology. Micro-CT results revealed that the rhBMP-2 group showed significantly higher calvarial defect coverage ratio compared with the ZA + rhBMP-2 group at 4 weeks. In addition, bone formation indices were significantly lower in ZA + rhBMP-2 group when compared with the rhBMP-2 group after 4 weeks, which indicates a negative effect of ZA on the initial bone formation and the bone quality. At 8 weeks, the negative effect induced by ZA treatment was alleviated as time passed. Histological examination showed similar results to the micro-CT measurements. In conclusion, although ZA treatment lowered the new bone formation induced by rhBMP-2 initially, as time passed, the negative effect was decreased.

Effects of Ovariectomy and Corticosteroid-Induced Osteoporosis on the Osteoinductivity of rhBMP-2 in a Segmental Long-Bone Defect Model.

This study used the segmental long-bone defect model to assess the effects of osteoporosis on the formation of new bones and the osteoinductivity of recombinant human bone morphogenetic protein-2 (rhBMP-2). Seventy-two female Sprague-Dawley rats were divided into two groups: an osteoporosis group with ovariectomies and dexamathasone intramuscular injections and a sham group. When they reached 22 weeks in age, each group was further divided into two groups and a 5-mm defect was made in both fibular mid-shafts of each rat. One fibula in each rat was picked randomly and was injected with 0.05 mL of hydrogel carrier; the opposite fibula was injected with the same carrier mixed with rhBMP-2 (10 µg). After rearing for a further 5 and 9 weeks, the ratios of the lengths of the newly formed bones in the fibular defects were determined using micro-CT and undecalcified histology. The sham rhBMP-2-injected group-in all of the 5- and 9-week-kept groups-showed a significantly higher bridging bone formation ratio than the other three groups. The osteoporosis rhBMP-2-injected group showed a significantly higher ratio than both the non-rhBMP-2-injected sham hydrogel and the osteoporosis hydrogel groups. The comparison of the micro-CT parameters of the newly formed bones showed that the sham rhBMP-2 group at both 5 and 9 weeks compared with the osteoporosis rhBMP-2 group had significantly higher percentage bone volumes, trabecular thicknesses, and trabecular numbers, in addition to significantly lower specific surfaces, trabecular pattern factors, and structural model indices. The histology results showed that the sham-rhBMP-2 group began forming bridging bones in the defect areas at 5 weeks, and at 9 weeks, trabeculae and marrow spaces were observed. However, the osteoporosis rhBMP-2 group exhibited a relatively minor level of new bone and trabecula formation. Consequently, the rhBMP-2 group showed significantly increased bone formation in the osteoporosis rat fibular defect model compared with the hydrogel group, whereas the new bone quantities, qualities, and remodeling in the osteoporosis rhBMP-2 group were less effective than those in the sham-rhBMP-2 group, signaling that ovariectomy and corticosteroid-induced osteoporosis significantly undermines rhBMP-2 osteoinductivity.

Enhanced osteoinductivity of recombinant human bone morphogenetic protein-2 in combination with epidermal growth factor in a rabbit tibial defect model.

This study aims to explore the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone formation when treated with epidermal growth factor (EGF) using human mesenchymal stem cells (hMSCs) and a rabbit tibial defect model. The rhBMP-2 (250 ng/ml)+EGF (10 ng/ml) group showed higher alkaline phosphatase (ALP) activity, ALP expression, increased calcium amount than rhBMP-2 group. In micro-CT and histology results of animal experiments, the rhBMP-2+EGF group showed more amount of bone bridging compared to the rhBMP-2 group. Among the 8-week groups, the rhBMP-2+EGF group showed significantly higher percent bone volume and trabecular number compared to the rhBMP-2 group. The combined treatment with EGF and rhBMP-2 induced significantly higher bone formation compared to that of rhBMP-2 only in both hMSCs and a rabbit tibial defect model. Therefore, EGF is expected to facilitate bone formation effect of rhBMP-2 when both factors are treated in combination.

Synergistic induction of early stage of bone formation by combination of recombinant human bone morphogenetic protein-2 and epidermal growth factor.

This study evaluates whether the combination of the rhBMP-2 and various types of growth factors including EGF, FGF, PDGF and VEGF increases osteoinductivity compared to the single use of rhBMP-2 through in vitro and in vivo study. Cultured human MSCs were treated with rhBMP-2 only or in combination with growth factors. For in vivo evaluation, rhBMP-2 only or with growth factors was implanted into the calvarial defect made on SD rats. Both EGF and PDGF significantly increased both ALP activity and expression level in hMSCs when treated in combination with rhBMP-2 at 3 and 7 days of differentiation and significantly raised the accumulation of the calcium at day 14. Furthermore, micro-CT scanning revealed that the EGF an FGF groups show significantly increased new bone surface ratio compared to the rhBMP-2 only group and, the EGF treatment significantly up regulated percent bone volume and trabecular number at two weeks after the surgery. VEGF treatment also significantly raised trabecular number and FGF treatment significantly increased the trabecular thickness. Histological examination revealed that the EGF combination group showed enhanced bone regeneration than the rhBMP-2 only group two weeks after the implantation. Even though the treatment of rhBMP-2 with PDGF and FGF failed to show enhanced osteogenesis in vitro and in vivo simultaneously, these results suggest that the positive effect of the combination of EGF and rhBMP-2 is expected to induce the bone formation earlier compared to the single use of rhBMP-2 in vitro and in vivo.

The effect of poloxamer 407-based hydrogel on the osteoinductivity of demineralized bone matrix.

BACKGROUND: Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water. METHODS: DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation. RESULTS: The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups. CONCLUSIONS: Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.

Effect of graphitic layers encapsulating single-crystal apatite nanowire on the osteogenesis of human mesenchymal stem cells.

An ideally designed scaffold for tissue engineering must be able to provide an environment that recapitulates the physiological conditions to control stem cell function. Here, we compared vertically aligned single-crystal apatite nanowires sheathed in graphitic layers (SANGs) with single-crystal apatite nanowires (SANs), which had the same geometric properties as--but differing nanotopographic surface chemistry than--SANGs, in order to evaluate the effect of the graphitic layer on the behavior of human mesenchymal stem cells (hMSCs). The difference in nanotopographic surface chemistry did not affect hMSC adhesion, growth, or morphology. However, hMSCs were more effectively differentiated into bone cells on SANGs through interaction with graphitic layers, which later degraded and thereby allowed the cells to continue differentiation on the bare apatite nanowires. Thus, SANGs provide an excellent microenvironment for the osteogenic differentiation of hMCS.

Generation of an rhBMP-2-loaded beta-tricalcium phosphate/hydrogel composite and evaluation of its efficacy on peri-implant bone formation.

Dental implant insertion on a site with low bone quality or bone defect should be preceded by a bone graft or artificial bone graft insertion to heal the defect. We generated a beta-tricalcium phosphate (ß-TCP) and poloxamer 407-based hydrogel composite and penetration of the ß-TCP/hydrogel composite into the peri-implant area of bone was evaluated by porous bone block experiments. The maximum penetration depth for porous bone blocks and dense bone blocks were 524 µm and 464 µm, respectively. We report the in-vivo performance of a composite of ß-TCP/hydrogel composite as a carrier of recombinant human bone morphogenetic protein (rhBMP-2), implanted into a rabbit tibial defect model. Three holes drilled into each tibia of eight male rabbits were (1) grafted with dental implant fixtures; (2) filled with ß-TCP/hydrogel composite (containing 5 µg of rhBMP-2), followed by grafting of the dental implant fixtures. Four weeks later, bone-implant contact ratio and peri-implant bone formation were analyzed by radiography, micro-CT and histology of undecalcified specimens. The micro-CT results showed a significantly higher level of trabecular thickness and new bone and peri-implant new bone formation in the experimental treatment compared to the control treatment. Histomorphometry revealed a significantly higher bone-implant contact ratio and peri-implant bone formation with the experimental treatment. The use of ß-TCP/poloxamer 407 hydrogel composite as a carrier of rhBMP-2 significantly promoted new bone formation around the dental implant fixture and it also improved the quality of the new bone formed in the tibial marrow space.

Fabrication of an rhBMP-2 loaded porous ß-TCP microsphere-hyaluronic acid-based powder gel composite and evaluation of implant osseointegration.

Methods to improve osseointegration that include implantation of rhBMP-2 with various kinds of carriers are currently of considerable interest. The present study was conducted to evaluate if the rhBMP-2 loaded ß-TCP microsphere-hyaluronic acid-based powder-like hydrogel composite (powder gel) can act as an effective rhBMP-2 carrier for implantation in host bone with a bone defect or poor bone quality. The release pattern for rhBMP-2 was then evaluated against an rhBMP-2-loaded collagen sponge as a control group. Dental implants were also inserted into the tibias of three groups of rabbits: an rhBMP-2 (200 µg) loaded powder gel composite implanted group, an implant only group, and a powder gel implanted group. Micro-CT and histology of the implanted areas were carried out four weeks later. The rhBMP-2 powder gel released less rhBMP-2 than the collagen sponge, but it continued a slow release for more than 7 days. The rhBMP-2 powder gel composite improved osseointegration of the dental implant by increasing the amount of new bone formation in the implant pitch and it improved the bone quality and bone quantity of new bone. The histology results indicated that the rhBMP-2 powder gel composite improved the osseointegration in the cortical bone as well as the marrow space along the fixture. The bone-to-implant contact ratio of the rhBMP-2 (200 µg) loaded powder gel composite implanted group was significantly higher than those of the implant only group and the powder gel implanted group. The powder gel appeared to be a good carrier and could release rhBMP-2 slowly to promote the formation of new bone following implantation in a bone defect, thereby improving implant osseointegration.

Magnetic manipulation of bacterial magnetic nanoparticle-loaded neurospheres.

Specific targeting of cells to sites of tissue damage and delivery of high numbers of transplanted cells to lesion tissue in vivo are critical parameters for the success of cell-based therapies. Here, we report a promising in vitro model system for studying the homing of transplanted cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres derived from human neuroblastoma SH-SY5Y cells labeled with bacterial magnetic nanoparticles were guided by a magnetic field and successfully accumulated near the focus site of the magnetic field. Our results demonstrate the effectiveness of using an in vitro model for testing bacterial magnetic nanoparticles to develop successful stem cell targeting strategies during fluid flow, which may ultimately be translated into in vivo targeted delivery of cells through circulation in various tissue-repair models.

Phosphorylated p70S6K in noninvasive low-grade urothelial carcinoma of the bladder: correlation with tumor recurrence.

We investigated whether inhibiting phosphorylated p70S6K (p-p70S6K) suppresses the proliferation and growth of noninvasive low-grade urothelial carcinoma (LG-URCa) in vitroand whether p-p70S6K can serve as a predictive biomarker for the recurrence of noninvasive LG-URCa of the bladder in patients. We constructed a tissue microarray (TMA) for 95 LG-URCa and 35 benign urothelium samples and performed immunohistochemical staining for p-p70S6K and p-4E-BP1. A Cox regression model was used to investigate the predictive factors for recurrence of LG-URCa. We investigated the dose-dependent antiproliferative effect of rapamycin, its antiproliferative effect and the growth-inhibition effect of p70S6K siRNA transfection in RT4 and 253J cell lines. The pT1 staged group (P < 0.05; hazard ratio (HR), 2.415) and the high p-p70S6K staining group (P < 0.05; HR, 2.249) were independent factors for predicting recurrence. Rapamycin inhibited RT4 and 253J cell proliferation in a dose-dependent manner (r = -0.850, P< 0.001 in RT4 cells; r = -0.835, P< 0.001 in 253J cells). RT4 and 253J cell proliferation and growth were inhibited by the transfection of p70S6K siRNA and rapamycin, respectively (P < 0.05). Transfection of p70S6K siRNA resulted in inhibitory effects on cell proliferation and growth that were similar to those of rapamycin. Our results suggest that inhibiting p70S6K phosphorylation is important to prevent recurrence and that p70S6K phosphorylation can be used as a molecular biomarker to predict recurrence of certain LG-URCa of the bladder.

The effects of recombinant human bone morphogenetic protein-2-loaded tricalcium phosphate microsphere-hydrogel composite on the osseointegration of dental implants in minipigs.

Bone formation in tooth defect areas and the osseointegration of dental implants are very important for successful dental implant surgery. The aim of the present study was to assess the strengthening effect of a ß-TCP microsphere-hydrogel composite containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone healing and implant osseointegration. The molars and premolars on the left and right sides of the maxilla were extracted from six male minipigs, and dental implants were placed using either the ß-TCP microsphere-hydrogel carrier alone or the carrier loaded with rhBMP-2 (500 µg). The animals were kept alive for a further 8 weeks. The molars and premolars from the left and the right sides of the mandibles of another six minipigs were extracted, and the animals were kept alive for 4 weeks. Two 5-mm-diameter bone defects were then made on both sides of the mandible. The defects were filled with saline, ß-TCP microsphere-hydrogel carrier, or the carrier loaded with rhBMP-2 (300 µg), and dental implant fixtures were inserted. The animals were kept alive for a further 4 weeks. Bone formation was examined using plane radiographs, micro-CT, and the histology of undecalcified specimens. The group treated with the rhBMP-2-loaded carrier composite showed a significantly higher percentage bone volume and a greater trabecular thickness for the newly formed bone in the tooth defect areas when compared to the group treated with the carrier alone. The rhBMP-2 group had a significantly higher osseointegration, a larger percentage bone volume, greater trabecular thickness in the newly formed bone in tooth defect areas, a larger newly formed bone fraction in the fixture pitch, and a greater number of newly formed trabecular bones when compared to the other groups. We confirmed that the rhBMP-2-loaded carrier composite promotes new bone formation after tooth extraction and strengthens osseointegration of dental fixtures by improving the degree of osseointegration around the dental implant fixture.