Effects of 1.8 GHz radiofrequency field on microstructure and bone metabolism of femur in mice.

To investigate the effects of 1.8 GHz radiofrequency (RF) field on bone microstructure and metabolism of femur in mice, C57BL/6 mice (male, age 4 weeks) were whole-body exposed or sham exposed to 1.8 GHz RF field. Specific absorption rates of whole body and bone were approximately 2.70 and 1.14 W/kg (6 h/day for 28 days). After exposure, microstructure and morphology of femur were observed by microcomputed tomography (micro-CT), Hematoxylin and Eosin (HE) and Masson staining. Subsequently, bone parameters were calculated directly from the reconstructed images, including structure model index, bone mineral density, trabecular bone volume/total volume, connectivity density, trabecular number, trabecular thickness, and trabecular separation. Biomarkers that reflect bone metabolism, such as serum total alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), and tartrate-resistant acid phosphatase 5b (TRACP-5b), were determined by biochemical assay methods. Micro-CT and histology results showed that there was no significant change in bone microstructure and the above parameters in RF group, compared with sham group. The activity of serum ALP and BALP increased 29.47% and 16.82%, respectively, in RF group, compared with sham group (P < 0.05). In addition, there were no significant differences in the activity of serum TRACP-5b between RF group and sham group. In brief, under present experimental conditions, we did not find support for an effect of 1.8 GHz RF field on bone microstructure; however, it might promote metabolic function of osteoblasts in mice. Bioelectromagnetics. 39:386-393, 2018. © 2018 Wiley Periodicals, Inc.

NPY and CGRP Inhibitor Influence on ERK Pathway and Macrophage Aggregation during Fracture Healing.

AIM: The aims of this study are to investigate the effects of neurotransmitters NPY and CGRP on ERK signaling in fracture healing, and to identify the correlation between macrophage aggregation and fracture healing. METHODS: Male Sprague-Dawley rats were used to build a fracture model. The neurotransmitter receptor inhibitors were injected intraperitoneally into the rats. Immunofluorescence staining and ELISA were employed to determine the expression of NPY and CGRP in fracture area and the peripheral blood, respectively. Micro-CT together with histological staining were utilized to assess the fracture healing conditions. Relative protein expression was determined using western blot. Immunofluorescence staining was used to detect the aggregation of macrophages in the injury area. RESULTS: During fracture healing, the serum NPY and CGRP significantly increased. The levels of NPY and CGRP reached a peak in the 8th week and reduced significantly thereafter. NPY and CGRP inhibitors could inhibit fracture healing and down-regulate the phosphorylated ERK. Macrophages (NPY+ and CGRP+) aggregated in the injury area. CONCLUSION: NPY and CGRP participated in fracture healing, in which they were also shown to influence phosphorylated ERK expression. In addition, macrophages are involved in the fracture healing process.

Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway.

Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5'-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.

Physical properties and biocompatibility of a core-sheath structure composite scaffold for bone tissue engineering in vitro.

Scaffolds play a critical role in the practical realization of bone tissue engineering. The purpose of this study was to assess whether a core-sheath structure composite scaffold possesses admirable physical properties and biocompatibility in vitro. A novel scaffold composed of poly(lactic-co-glycolic acid)/ß-tricalcium phosphate (PLGA/ß-TCP) skeleton wrapped with Type I collagen via low-temperature deposition manufacturing (LDM) was prepared, and bone mesenchymal stem cells (BMSCs) were used to evaluate cell behavior on the scaffold. PLGA/ß-TCP skeleton was chosen as the control group. Physical properties were evaluated by pority ratio, compressive strength, and Young's modulus. Scanning electron microscope (SEM) was used to study morphology of cells. Hydrophilicity was evaluated by water absorption ratio. Cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT). Osteogenic differentiation of BMSCs was evaluated by alkaline phosphates activity (ALP). The results indicated that physical properties of the novel scaffold were as good as those of the control group, hydrophilicity was observably better (P < 0.01) than that of control group, and abilities of proliferation and osteogenic differentiation of BMSCs on novel scaffold were significantly greater (P < 0.05) than those of control group, which suggests that the novel scaffold possesses preferable characteristics and have high value in bone tissue engineering.

Repair of distal fibular and lateral malleolus defects with individualized 3D-printed titanium alloy prosthesis: The first case report from China.

INTRODUCTION AND IMPORTANCE: This case report describes the reconstruction of the traumatic distal fibular and lateral malleolus defects with a novel method of using individualized 3D printed titanium prosthesis for the first time. CASE PRESENTATION: A 63-year-old male farmer was hospitalized in emergency because of open injury and distal fibular and lateral malleolus defects in the left leg caused by a car accident. 3 months after debridement and latissimus dorsi muscle flap transplantation and skin graft operation, the patient re-hospitalized because of distal fibular and lateral malleolus defect and local pain. We examined the bilateral ankle joint with three-dimensional CT, obtained data about the missing left distal fibular and lateral malleolus through the mirror principle. The corresponding titanium alloy prosthesis then was designed and printed using a 3D metal printer. The patient had no obvious contraindication for surgery, so the prosthesis was surgically implanted. The patient was followed up for 2 years. There was no discomfort at the surgical site. The function of the operated ankle was satisfied by the patient, the AOFAS (American Orthopaedic Foot & Ankle Society) score was 85 (Kitaoka et al., 1994 [1]). CLINICAL DISCUSSION: Individualized 3D printed titanium alloy prosthesis consistent with the anatomical structure of lost distal fibula and lateral malleolus. The proximal end of the prosthesis was designed with four nail holes to install screws to fix the fibula together with it. The lower tibiofibular and talofibular joint surfaces of the prosthesis were designed smoothly. In order to improve the stability of the lower tibiofibular joint, anchors were placed at the attachment of the anterior and posterior tibiofibular ligaments to reconstruct these ligaments. CONCLUSION: The structure and function of the reconstructed distal fibular and the lateral malleous were close to normal. Individualized 3D printed prosthesis might have considerable advantages over traditional treatment methods. The individualized 3D printed titanium alloy prosthesis provides a new method for the repair and reconstruction of similar bone defects. The use of 3D printed prosthesis for surgical repair needs to be further examined in the future through long-term follow-up studies and in more cases.

Repair of segmental ulnar bone defect in juvenile caused by osteomyelitis with induced membrane combined with tissue-engineered bone: A case report with 4-year follow-up.

INTRODUCTION AND IMPORTANCE: We used induced membrane combined with tissue-engineered bone (TEB) to repair the 14-cm juvenile ulnar defect formed after osteomyelitis debridement. The TEB was completely transformed into autologous bone after 4-year follow-up. CASE PRESENTATION: A 13-year-old male was hospitalized because of right ulna chronic osteomyelitis. After focal debridement, the total length of ular defect was 14 cm. Anti-infective bone cement was filled in the bone defect area. ß-Tricalcium phosphate (ß-TCP) was used as TEB scaffold. Autologous iliac bone marrow stromal cells (BMSCs) were cultured in vitro and were planted on ß-TCP scaffold to form TEB 3 weeks later. 47 months after implantation of TEB, the repaired ulna had continuous and smooth bone cortex, completely ossification of TEB, completely recanalization of medullary cavity. The upper limb function DASH score was 35. CLINICAL DISCUSSION: Masquelet put forward the concept of "induced membrane" and applied this technique on bone defects treatment formed after debridement of osteomyelitis. ß-Tricalcium phosphate (ß-TCP) is artificial bone materials commonly used in clinical. In this case, the seed cells used were autologous BMSCs and the culture medium was autologous serum. Cytokines promoting cell growth and differentiation were not used. CONCLUSION: The results of this case showed that TEB combined with induced membrane could repair ulna segmental bone defects as long as 14 cm in adolescents. This technique gives one alternative method to repair juvenile bone defects caused by osteomyelities of trauma. More clinical cases are needed to verify the effectiveness of this technique in the next.

Mineral status and mechanical properties of cancellous bone exposed to hydrogen peroxide for various time periods.

Processed cancellous bone has been regarded as one alternative for the treatment of bone defects. In order to avoid immunogenic effects and preserve the natural properties of the bone, the optimal processing method should be determined. To observe the influence of hydrogen peroxide on the mineral status and mechanical properties of cancellous bone for various time periods and find the optimal processing time. Cancellous bone granules from bovine femur condyles were treated with 30% hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 h separately. The microstructure and mineral content of the granules were evaluated by ash analysis, Micro-CT, scanning electron micrograph and energy dispersive X-ray. The biomechanical properties were analyzed by applying cranial-caudal compression in a materials testing machine. With increasing exposure to hydrogen peroxide, the BMD and BMC of granules gradually decreased, and the Ca/P molar ratios clearly increased (P < 0.05). Meanwhile, the mineral content of the granules increased from 48.5 ± 1.3 to 79.5 ± 2.1%. Substantial decreases in the strength of the granules were observed, and after 48 h severe decreases were noted. The decrease in strength was also evident after normalizing the parameters to the cross-sectional area. Granules of bovine cancellous bone matrix should be processed by hydrogen peroxide for 12 to 36 h to fulfill the basic requirements of a bone tissue engineering scaffold. These granules could potentially be useful during orthopedic operations.

Multi-variety bone bank in China.

This is a descriptive report of the establishment and operation of a Chinese bone bank, though not a typical one. While being engaged in collection, processing and storage of allogeneic tissues, the bone bank to which the author belongs concurrently develops and produces new, non-human derived, graft materials. Among others is reconstituted bone xenograft (RBX) which possesses strong osteoinductive capability without evoking immune rejection. Hence, its appellation "multi-variety bone bank," which was established by Dr. Hu Yunyu in 1990, the first of its kind in China. There are several salient features discriminating this bone bank from others. At this hospital-based non-profit institution, allograft hemi-joints are freshly prepared and distributed deep-frozen, instead of being freeze-dried on an industrialized basis for convenient transportation. The former has much more superior biological and mechanical properties as compared with the latter. However, allogeneic tissues are sometimes in short supply due to limited number of donors and the risk of some potential donors carrying viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV). New graft materials, including reconstituted bone xenograft (RBX), were developed that serve as a supplement to allografts. RBX has been successfully used in clinical practice for the management of old fractures, nonunions and bone defects, most notably of contaminated, infected open fractures and osteomyelitis with the use of anti-infective reconstituted bone xenograft (ARBX). Additionally the multi-variety bone bank serves as a training base for educating professional personnel and researchers (postgraduates) in theories and technologies of tissue banking. Up to now, eighteen special technical staff members and approximately sixty senior researchers have been trained at this institution.

Lanthanides-Substituted Hydroxyapatite/Aloe vera Composite Coated Titanium Plate for Bone Tissue Regeneration.

PURPOSE: To develop the surface-treated metal implant with highly encouraged positive properties, including high anti-corrosiveness, bio-activeness and bio-compatibleness for orthopedic applications. METHODS: In this work, the surface of commercially pure titanium (Ti) metal was treated with bio-compatible polydopamine (PD) by merely immersing the Ti plate in PD solution. The composite of trivalent lanthanide minerals (La3+, Ce3+ and Gd3+)-substituted hydroxyapatite (MHAP) with Aloe vera (AV) gel was prepared and coated on the PD-Ti plate by electrophoretic deposition (EPD) method. The choice of trivalent lanthanide ions is based on their bio-compatible nature and bone-seeking properties. The formation of the PD layer, composites, and composite coatings on Ti plate and PD-Ti surface was confirmed by FT-IR, XRD, SEM and HR-TEM observations. In-vitro assessments such as osteoblasts like MG-63 cell viability, alkaline phosphatase activity and mineralization ability of the MHAP/AV composite were tested, and the composite-coated plate was implanted into a rat bone defect model for in-vivo bone regeneration studies. RESULTS: The coating ability of the MHAP/AV composite was highly preferred to PD-treated Ti plate than an untreated Ti plate due to the metal absorption ability of PD. This was confirmed by SEM analysis. The in-vitro and in-vivo studies show the better osteogenic ability of MHAP/AV composite at 14th day and 4th week of an experimental period, respectively. CONCLUSION: The osteoblast ability of the fabricated device without producing any adverse effect in the rat model recommends that the fabricated device would serve as a better platform on the hard tissue regeneration for load-bearing applications of orthopedics.

Treatment of contaminated bone defects with clindamycin-reconstituted bone xenograft-composites.

Contaminated or infected bone defects and osteomyelitis after trauma are frequently encountered in clinical practice. It is difficult to make a successful bone graft and control infection at the same time. To find a better method to resolve this dilemma, we prepared a novel clindamycin-reconstituted bone xenograft-composite (C-RBX-C) that comprised of crude bBMP (bovine bone morphogenetic protein), clindamycin, and cancellous bone scaffold, and investigated the morphology, biocompatibility, antibiotic release profile and osteoinductive potential of this composite. The ultrastructure of C-RBX-C was evaluated by scanning electron microscopy; the biocompatibility and osteoinductive potential were assessed by testing ectopic implants. The antibiotic release profile was evaluated using a disc-diffusion assay. Finally, this composite was used to repair a Staphylococcus aureus contaminated bone defect in a rabbit model. 16 weeks after the implantation of C-RBX-C, the radial defect had been completely recuperated, with significantly better formation of lamellar bone and recanalization of the marrow cavity, than in the controls (scaffolds without clindamycin or bBMP). These results demonstrate that our novel composite, with its concomitant osteoinductive and antibiotic properties, has significant potential for the treatment of contaminated or infected bone defects and osteomyelitis.

[Three dimensional induction of autologous mesenchymal stem cell and the effects on depressing long-term degeneration of tissue-engineering cartilage].

OBJECTIVES: To induce autologous bone marrow derived mesenchymal stem cell (aMSC) into chondrocyte, and to confirm the effects of 3 dimensional (3D) dynamic inducing in vitro and their long-term animal model repairing in vivo. METHODS: aMSC were separated from rabbits bone marrow aspirates, then respectively experienced 3D dynamic inducing in alginate drops in modified rotating wall bioreactor culture or in two dimensional (2D) inducing (culture flask) for 10 d. The induced cells were harvest and then mixed with fibrin sealant (FS) to repair rabbit knee femoral trochlea cartilage defects model. After 8, 12, 24, 48 weeks animals were euthanized. Gross appearance, histological appearances were examined. RESULTS: Flask culture groups showed a little chondrocyte differentiation, 3D inducing group showed obviously chondrocyte differentiation, improved collagen II and proteoglycan production. For 3D inducing ones in vivo, the cartilage defects were smoothly repaired by white translucent hard tissue with obvious hyaline-like cartilage histological appearance after 8, 12 weeks, and the defects boundary were hard to be identified with hyaline like cartilage with sustained histological appearance and score after 24, 48 weeks. For 2D ones in vivo, the cartilage defects were smoothly repaired after 8 weeks by hyaline like cartilage which showed accelerated degeneration after 24 weeks and lose cartilage performance completely after 48 weeks. CONCLUSIONS: 3D dynamic inducing may assist aMSC on differentiating into chondrocyte, improve its long-term in vivo repairing effects, and enlighten its further applications in tissue engineering cartilage.

Expression profile of cytokines and chemokines in osteoarthritis patients: Proinflammatory roles for CXCL8 and CXCL11 to chondrocytes.

There are interactions between immune response and destruction of articular cartilage/synovial tissue in osteoarthritis (OA), which leads to chronic inflammation and systemic failure of joints. However, the role of immunological factors in the pathogenesis of OA has not been fully elucidated. In this study, expressions of 47 cytokines and chemokines were tested in the peripheral bloods and synovial fluids from 13 normal controls (NCs) and 31 OA patients. The primary chondrocytes, which were isolated from cartilages of OA patients, were stimulated by recombinant CXCL8 and CXCL11 to analyze the proliferation, cytokine secretion, and signaling pathways. The levels of IL-17A, CXCL8, CXCL9, and CXCL11 were elevated in the serum and synovial fluids of OA patients. Moreover, expressions of CXCL8 and CXCL11 were remarkably increased in the synovial fluids of late stage OA. Stimulation of CXCL8/11 resulted in the reduction of primary chondrocytes proliferation with downregulation of G2-M stage but elevation of S stage and apoptosis cells. The secretions of proinflammatory cytokines and MMPs were also increased upon stimulation. Furthermore, CXCL8/11 stimulation induced the higher expressions of phosphorylated STAT3, NF-kB p50 and JNK, but not p38MAPK or ERK1/2. Our findings suggested that CXCL8 and CXCL11 promoted the apoptosis and suppressed the proliferation of chondrocytes probably via influencing JAK-STAT, NF-kB and JNK MAPK signaling pathway and enhancing the expressions of other proinflammatory cytokines. CXCL8/11 may aggravate the disease progression of OA, and may also be served as new therapeutic targets for treatment of OA.

Effects of local delivery of BMP2, zoledronate and their combination on bone microarchitecture, biomechanics and bone turnover in osteoporotic rabbits.

The hip fracture is one major clinical challenge associated with osteoporosis, resulting in heavy socioeconomic burdens and high mortality. Systemic therapies of anti-osteoporosis drugs are expensive, time-consuming and also evoke substantial side effects, which fails to provide early protection from fractures. Accumulating evidence demonstrates the high bioavailability and therapeutic efficacy of local drug delivery in accelerating facture healing and bone defect repair. This study aims at investigating the effects of local delivery of BMP2 and zoledronate (two promising anabolic/anti-catobolic reagents) encapsulated by fibrin sealants into femoral necks on regulating bone quality and remodeling in osteoporotic rabbits subjected to combined ovariectomy and glucocorticoid injection. We show that 6-week BMP2 delivery exhibited more prominent effect on mitigating trabecular bone microarchitecture deterioration and mechanical strength reduction of femoral necks than local zoledronate treatment. BMP2 plus zoledronate showed more significant improvement of bone microstructure, mechanical strength and bone formation rate at 12 weeks post injection than single BMP2 or zoledronate delivery via µCT, biomechanical, histomorphometric and serum biochemical analyses. This study enriches our knowledge for understanding the availability of local drug delivery for improving bone quantity and quality, which may lead to earlier, safer and more efficient protection from osteoporosis-induced fractures in clinics.

[Morphology research of the rat sciatic nerve bridged by collage-heparin sulfate scaffold].

OBJECTIVE: To observe the treating effect of collage-heparin sulfate after the 10 mm rat sciatic nerve defect was bridged by it. METHODS: A new kind of nervous tissue engineering scaffold was produced by freeze-drying technique from collagen-heparin sulfate. Thirty-two SD rats were randomly divided into A, B, C and D groups. Sciatic nerve defect in group A was bridged by collagen-heparin sulfate. In group B, sciatic nerve was bridged by auto-nerve transplantation. Group C was the blank control group. Animals in group D were normal. And 10 mm sciatic nerve defect was bridged in the experiment. Thirty-six weeks after the operation, the experimental animals were detected by HRP labeled retrograde trace, HE staining, toluidine staining, silvering staining, S100, GAP-43 and NF immunohistological staining, MBP immunofluorescence staining and transmission electron microscope to observe the nerve regeneration inducing effect of this new scaffold. RESULTS: Nine months after operation, the collage-heparin sulfate scaffold was replaced by newly regenerated nerve. The number of HRP labeled spinal cord anterior horn cells and the area of sensation nerve fiber at the posterior horn were similar with that was repaired by auto-nerve. GAP-43, NF and S100 labeled regenerated nerve fiber had passed the total scaffold and entered the distal terminal. The regenerated nerve fibers were paralleled, lineage arranged, coincide with the prearranged regenerating "channel" in the collagen-heparin sulfate scaffold. MBP immunofluorescence staining also proved that the newly regenerated nerve fiber could be ensheathed. In the experimental group, the area of myelinated nerve fiber and the thickness of the myelin sheath had no obvious difference with that of the group repaired by auto-nerve, except that the density of the regenerated myelinated sheath fiber was lower than that of the control group. CONCLUSION: Nervous tissue engineering scaffold produced by collagen-heparin sulfate can guide the regeneration of nerve fibers. The nerve function recovers fine. This kind of material has great application potential.

MicroRNA­20a promotes the proliferation and cell cycle of human osteosarcoma cells by suppressing early growth response 2 expression.

MicroRNAs (miRNAs) are crucial in cancer development. However, the underlying mechanisms of miRNAs in osteosarcoma (OS) remain largely uncharacterized. The present study investigated the role of miR­20a in OS cell proliferation. It was determined that miR­20a expression is markedly upregulated in OS tissues and cells compared with the matched adjacent normal tissues and h­FOB human osteoblast cell lines. Ectopic expression of miR­20a promoted the proliferation and anchorage­independent growth of OS cells, whereas inhibition of miR­20a reduced this effect. Bioinformatics analysis further revealed early growth response 2 (EGR2), as a potential target of miR­20a. Data from luciferase reporter assays showed that miR­20a directly binds to the 3'­untranslated region (3'­UTR) of EGR2 mRNA and represses expression at the transcriptional and translational levels. In functional assays, miR­20a promoted OS cell proliferation and the cell cycle, which could be suppressed by an inhibitor of miR­20a. In conclusion, the data provide compelling evidence that miR­20a functions as an onco­miRNA, which is important in promoting cell proliferation in OS, and its oncogenic effect is mediated primarily through direct suppression of EGR2 expression.

Treatment of tibial defect and bone nonunion with limb shortening with external fixator and reconstituted bone xenograft.

OBJECTIVE: To explore the effect of external fixator and reconstituted bone xenograft (RBX) in the treatment of tibial bone defect, tibial bone nonunion and congenital pseudarthrosis of the tibia with limb shortening. METHODS: Twenty patients (13 males and 7 females) with tibial bone defect, tibial bone nonunion or congenital pseudarthrosis of the tibia with limb shortening were treated with external fixation. Two kinds of external fixators were used: a half ring sulcated external fixator used in 13 patients and a combined external fixator in 7 patients. Foot-drop was corrected at the same time with external fixation in 4 patients. The shortened length of the tibia was in the range of 2-9 cm, with an average of 4.8 cm. For bone grafting, RBX was used in 12 patients, autogenous ilium was used in 3 patients and autogenous fibula was implanted as a bone plug into the medullary canal in 1 case, and no bone graft was used in 4 patients. RESULTS: All the 20 patients were followed-up for 8 months to 7 years, averaging 51 months. Satisfactory function of the affected extremities was obtained. All the shortened extremities were lengthened to the expected length. For all the lengthening area and the fracture sites, bone union was obtained at the last. The average healing time of 12 patients treated with RBX was 4.8 months. CONCLUSIONS: Both the half ring sulcated external fixator and the combined external fixator have the advantages of small trauma, simple operation, elastic fixation without stress shielding and non-limitation from local soft tissue conditions, and there is satisfactory functional recovery of affected extremities in the treatment of tibial bone defects, tibial bone nonunion and congenital pseudarthrosis of the tibia combined with limb shortening. RBX has good biocompatibility and does not cause immunological rejections. It can also be safely used in treatment of bone nonunion and has reliable effect to promote bone healing.

Reciprocal action between BMP-2 and BMP-3 in cultured fibroblast in vitro.

OBJECTIVE: To explore reciprocal action between BMP-2 (bone morphogenetic protein-2) and BMP-3 for better understanding of the mechanism of BMP during bone fracture union. METHODS: rhBMP-2 was added into the cultured fibroblasts with the concentration of 1,200 ng/ml. The expression of BMP-3 in fibroblasts was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3-BMP-3 was transfected into the fibroblasts. After the effective expression of BMP-3 was identified, BMP-2 was also detected by immunohistochemistry in BMP-3 expression cells. The fibroblasts transfected with empty vector pcDNA3 were used as the control. RESULTS: Exogenous rhBMP-2 could promote the expression of BMP-3 in fibroblasts. BMP-3 also could be detected in these cells. CONCLUSIONS: BMP-2 and BMP-3 could reciprocally adjust the expression in fibroblasts.

Adenovirus-mediated transfer of VEGF into marrow stromal cells combined with PLGA/TCP scaffold increases vascularization and promotes bone repair in vivo.

INTRODUCTION: Large osseous defect remains a serious clinical problem due to the lack of sufficient blood supply and it has been proposed that this situation can be relieved by accelerating the formation of new vessels in the process of bone defect repair. The aim of this study was to develop a new type of artificial bone by transferring the VEGF gene into marrow stromal cells (MSCs) and seeding them into a porous scaffold. MATERIAL AND METHODS: An adenovirus vector was employed to transfer the VEGF gene into MSCs and expression of the exogenous gene was confirmed by ELISA. Next the transduced cells were seeded into a collagen I modified PLGA/TCP scaffold. The constructed new complex artificial bone was then assessed for biocompatibility in vitro and blood vessel formation and bone formation in vivo. RESULTS: We found that adenovirus mediated VEGF gene transfer into MSCs sustained VEGF expression in MSCs for 3 weeks. Porous scaffold PLGA/TCP made by rapid prototyping technology exhibited improved biocompatibility resulting from crosslinking with collagen I. Furthermore, the in vivo study showed that large amounts of blood vessels were detected histologically 1 week after artificial bone implantation, and significant bone formation was detected 8 weeks after implantation. CONCLUSIONS: Our findings suggest that gene transfer of VEGF into MSCs combined with PLGA/TCP scaffold enhances bone repair in vivo by promoting vascularization.

FFAs-ROS-ERK/P38 pathway plays a key role in adipocyte lipotoxicity on osteoblasts in co-culture.

The accumulation of adipocytes in bone marrow is common in a variety of pathophysiological conditions, including obesity, insulin resistance, type 2 diabetes, and aging. Adipocytes in bone marrow exhibit severe adverse effect on osteoblast differentiation, proliferation, and function. However, the molecular mechanism of adipocytes lipotoxicity on osteoblasts is still far from completely understood. The present study was designed to investigate the signaling pathway responsible for adipocytes lipotoxicity on osteoblasts. Using a co-culture system, we have identified that free fatty acids (FFAs) released by the adipocytes inhibited osteoblasts proliferation and function and induced osteoblasts apoptosis, evidenced by decreased cell viability/proliferation, ALP activity, expression of runt-related transcription factor 2 (RunX2), type I collagen (ColA1) and osteocalcin and alizarin red staining. Dexamethasone (Dex) promoted the inhibitory effect of adipocytes on osteoblasts through stimulating FFAs release. Dex-exacerbated FFAs release from adipocytes contributes to reactive oxygen species (ROS) production. In the co-culture system, the phosphorylation of extracellular signal-regulated kinase (ERK)/P38 was increased and inhibition of ERK/P38 significantly suppressed adipocytes lipotoxicity. FFAs-generated ROS was responsible for adipocytes-induced activation of ERK/P38 signaling. In conclusion, FFAs-ROS-ERK/P38 pathway plays a key role in adipocyte lipotoxicity on osteoblasts in co-culture. The evidence provides new insights into the mechanisms underlying the lipotoxic effect of adipocytes on bone within the marrow microenvironment and prevention of lipotoxicity on bone metabolism.

MicroRNA­125b suppresses the proliferation and osteogenic differentiation of human bone marrow­derived mesenchymal stem cells.

The regressive biological function of human bone marrow­derived mesenchymal stem cells (hBMSCs) is one of the key factors resulting in the decrease of bone mass in senile osteoporosis. MicroRNAs (miRs) are non­coding small RNAs involved in various gene regulation processes. Whether any miR(s) are involved in the progression of osteoporosis by regulating the biological function of hBMSCs remains to be elucidated. The present study aimed to compare the expression levels of miR­125b in hBMSCs derived from senile osteoporotic patients with that of control (normal) subjects. A significantly upregulated expression of miR­125b in osteoporotic hBMSCs was detected. To elucidate the biological function of miR­125b in senile osteoporosis, the effects of miR­125b expression on proliferation and osteogenic differentiation of hBMSCs were assessed using gain­ and loss­of­function studies. It was evident that the overexpression of a miR­125b mimic was able to suppress the proliferative and osteogenic differentiation of senile hBMSCs. In contrast, repression of the function of miR­125b by transfection of an miR­125b inhibitor promoted the proliferation and osteogenic differentiation of hBMSCs. Furthermore, the potential target gene of miR­125b, osterix (Osx), was examined. The results of the present study strongly suggested that miR­125b may regulate osteogenic differentiation of hBMSCs through the modulation of Osx expression.