Bone Marrow Formation Research Articles

Exercise Promotes the Osteoinduction of HA/β-TCP Biomaterials via the Wnt Signaling Pathway

To investigate the osteoinductive mechanism triggered by hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) biomaterials in mice which keep exercising. Methods: The HA/β-TCP biomaterials were implanted in the muscle of bilateral thighs (non-osseous sites) of eighty Balb/C mice. All animals were then randomly divided into 4 groups (n = 20). In group 1 (negative control group), the mice were fed routinely. In group 2 (running group), all mice were put on a treadmill which was set to a 60-degree incline. The mice ran 20 min thrice each day. A 5-minute break was included in the routine from day three onwards. In group 3 (weight-bearing group), all mice underwent weight-bearing running. The mice in this group performed the same routine as group 2 while carrying 5 g rubber weights. In group 4 (positive control group), dexamethasone was injected in the implanted sites of the biomaterials from the day of the operation. All mice were injected once per week and received a total of 8 injections. One and eight weeks after surgery, the blood serum was collected to detect inflammatory and immunological factors by ELISA. In addition to this, biomaterial specimens were obtained to observe inflammatory and osteogenic levels via histological staining and to facilitate analysis of the osteogenic mechanism by Western Blot. Results: The inflammation indexes caused by surgery were alleviated through running or weight-bearing running: The tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were significantly reduced in groups 2 and 3 at week 8. Exercise also enhanced the secretion of interferon-γ (IFN-γ) in mice; this can strengthen their immunity. The new bone tissues were observed in all groups; however, the area percentage of new bone tissues and the number of osteoblasts were highest in the weight-bearing group. Furthermore, the key proteins of wingless/integrated (Wnt) signaling pathway, Wnt1, Wnt3a, and β-catenin, were up-regulated during osteoinduction. This up-regulation activated runt-related transcription factor-2 (Runx2), increased the expression of osteopontin (OPN) and osteocalcin (OCN). Conclusion: Weight-bearing exercise can promote the bone and bone marrow formation through the Wnt signaling pathway: Observations documented here suggest that the proper exercise is beneficial to the recovery of bone damage.

Extracellular vesicles secreted from mouse muscle cells suppress osteoclast formation: Roles of mitochondrial energy metabolism.

Recent reports have described the interactions of muscle and bone. Various muscle-derived humoral factors, known as myokines, affect bone. Although extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring their contents to distant tissues during bone metabolism, the roles of EVs in the muscle-bone interactions remain unknown. In the present study, we investigated the effects of EVs secreted from mouse muscle C2C12 cells on mouse bone cells and mitochondrial biogenesis. EVs secreted from C2C12 cells (Myo-EVs) were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation as well as the expression of tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cells cytoplasmic 1 and dendritic cell-specific transmembrane protein induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bone marrow cells and preosteoclastic Raw264.7 cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expression of the mitochondrial biogenesis markers enhanced by RANKL in these cells. However, Myo-EVs did not affect the phenotypes or mitochondrial biogenesis of mouse primary osteoblasts. In conclusion, the present study showed for the first time that Myo-EVs suppress osteoclast formation and mitochondrial energy metabolism in mouse bone marrow and Raw264.7 cells. EVs secreted from skeletal muscles might be a crucial mediator of muscle-bone interactions.

Improved physical and osteoinductive properties of demineralized bone matrix by gelatin methacryloyl formulation.

The demineralized bone matrix (DBM) is the most widely used bone allograft, which is obtained by removing the mineral component of bone, leading to exposure of the proteins responsible for osteoinduction. For clinical use, DBM shall be formulated with a carrier that provides consistency and improves its osteoinduction. In this study, three DBM formulations with glycerol (Gly), hyaluronic acid (HA), and gelatin methacryloyl (GelMA) were evaluated measuring their physicochemical properties (microstructure, compressive strength, and serum cohesivity) and their osteoinductive capacity both in vitro using C2C12 cells and umbilical cord human mesenchymal stem cells and in vivo in an ectopic bone formation model in athymic mice. To assess the effectiveness of DBM in vitro in inducing the differentiation into osteoblasts, alkaline phosphatase (ALP) activity was assessed in combination with a cytotoxicity assay. In vivo, new bone formation was assessed by histological and radiological analysis. In the compression and in the serum cohesive assays, the GelMA DBM formulation showed its superiority over the other formulations. In addition, GelMA showed a more compact structure analysed by scanning electron microscopy. Higher cell toxicity was observed on Gly formulations in vitro, whereas GelMa and HA showed very low toxicity. All formulations significantly improved ALP activity compared with control. In the in vivo studies, GelMA showed the greatest osteoinductive potential with a higher percentage of new bone and bone marrow formation. Our results suggest GelMA is useful as a carrier for DBM designed to promote the formation of the new bone.

Dispersion of ceramic granules within human fractionated adipose tissue to enhance endochondral bone formation

Engineering of materials consisting of hypertrophic cartilage, as physiological template for de novo bone formation through endochondral ossification (ECO), holds promise as a new class of biological bone substitutes. Here, we assessed the efficiency and reproducibility of bone formation induced by the combination of ceramic granules with fractionated human adipose tissue (“nanofat”), followed by in vitro priming to hypertrophic cartilage. Human nanofat was mixed with different volumetric ratios of ceramic granules (0.2-1 mm) and cultured to sequentially induce proliferation (3 weeks), chondrogenesis (4 weeks), and hypertrophy (2 weeks). The resulting engineered constructs were implanted ectopically in nude mouse. The presence of ceramic granules regulated tissue formation, both in vitro and in vivo. In particular, their dispersion in nanofat at a ratio of 1:16 led to significantly increased cell number and glycosaminoglycan accumulation in vitro, as well as amount and inter-donor reproducibility of bone formation in vivo. Our findings outline a strategy for efficient utilization of nanofat for bone regeneration in an autologous setting, which should now be tested at an orthotopic site. Statement of significanceIn this study, we assessed the efficiency and reproducibility of bone formation by a combination of ceramic granules and fractionated human adipose tissue, also known as nanofat, in vitro primed into hypertrophic cartilage. The resulting engineered cartilaginous constructs, when implanted ectopically in nude mouse, resulted in bone and bone marrow formation, more reproducibly and strongly that nanofat alone. This project evaluates the impact of ceramic granules on the functionality and chondrogenic differentiation of mesenchymal progenitors inside their native adipose tissue niche and outlines a novel strategy for an efficient application of nanofat for bone regeneration in an autologous setting.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Toll-like receptor 4 protects against irradiation-induced hematopoietic injury by promoting granulopoiesis and alleviating marrow adipogenesis

Irradiation induces severe damage in the hematopoietic system, which leads to bone marrow hyperplasia, pancytopenia, and aggravated tissue formation in bone marrow. Studies have shown that Toll-like receptor 4 (TLR4) has a protective effect against irradiation, but the underlying mechanism remains unclear. In this study, we used a TLR4 knockout (TLR4−/−) mouse irradiation model and found that the white blood cell and platelet counts in the peripheral blood of TLR4−/− mice recovered slowly after irradiation, with bone marrow hyperplasia and increased mortality. Additionally, we found that the proportion of CD11b+Gr1+ granulocytes in the peripheral blood and bone marrow of TLR4−/− mice was lower than that of wild-type mice after irradiation. Further, we found that the expression of NADPH Oxidases (NOXs) in the bone marrow was down-regulated after irradiation of TLR4−/− mice, and administration of the NOXs inhibitor VAS2870 reduced the proportion of CD11b+Gr1+ cells in the bone marrow and peripheral blood of wild-type mice after irradiation. Irradiation induced severe marrow adipocytes accumulation in TLR4−/− mice, TLR4 ligand lipopolysaccharide promoted proliferation and inhibited adipogenic differentiation of mesenchymal stromal cells. In summary, our data suggest that TLR4 promotes myeloid hyperplasia by up-regulating the expression of NOXs after irradiation, prohibits marrow adipogensis and increases the tolerance of mice to irradiation.

Carbonate Apatite Micro-Honeycombed Blocks Generate Bone Marrow-Like Tissues as well as Bone.

Hematopoietic stem cells form blood cells in bone marrow and reside in niches. Artificial environments that conserve these niches may generate bone marrow. Osteogenesis, angiogenesis, and material resorption must be regulated to create these environments. These processes are controlled by material composition and macro- and microporous structures. Here, three blocks with different micropore structures are fabricated. Carbonate apatite has nearly the same composition as natural human bone and their honeycomb structure facilitates cell penetration and survival. In samples with high microporosity, endosteum-like tissues such as sinusoids form in areas of material resorption and high local calcium concentration. These conditions resemble environments conducive to niche maintenance. Bone marrow-like tissues and megakaryocytes are successfully generated in this environment. Micropore structure is the most critical factor in bone marrow formation; however, the influences of material composition and macropore structure must also be considered. The results of this study may help develop treatments for bone marrow-related diseases and elucidate the components and functions of the hematopoietic stem cell niche.

TGFβ signaling underlies hematopoietic dysfunction and bone marrow failure in Shwachman-Diamond Syndrome.

Shwachman-Diamond Syndrome (SDS) is a rare and clinically-heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. Although SDS was described over 50 years ago, the molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from SDS patients. We generated a single cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor-beta (TGFβ) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGFβ inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGFβ3 and other TGFβ pathway members were elevated in SDS patient blood plasma. These data establish the TGFβ pathway as a novel candidate biomarker and therapeutic target in SDS and translate insights from single cell biology into a potential therapy.

Hepatotoxicity and chromosomal abnormalities evaluation due to single and repeated oral exposures of chromium oxide nanoparticles in Wistar rats.

Metal oxide nanoparticles (NPs) have widespread uses ranging from nanoelectronics to nanotherapeutics. Because of their expanding industrial applications, a better understanding of their toxicity is needed. So far, limited reports are available on chromium oxide NPs (Cr2O3 NPs) toxicity. In this work, Cr2O3 NPs were synthesized and characterized in a sequential manner using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy. Dose- and time-dependent toxicity assessment of Cr2O3 NPs was carried out in Wistar rats by examining liver function biomarkers, tissue histopathology, micronuclei (MN) formation, and chromosomal aberrations (CAs) in bone marrow along with sperm abnormalities. The results of this study demonstrated typical XRD and FTIR patterns of Cr2O3 NPs with a size of approximately 23.47 nm. Animals exposed to Cr2O3 NPs, exhibited a significant increase in aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyltransferase, and total bilirubin, signifying liver injury. Histopathology data also supported the marked alterations in the liver biochemistry of NPs-exposed animals. Further, an increase in the frequency of MN, CA, and sperm abnormalities suggested Cr2O3 NPs-mediated genotoxicity. It is, therefore, suggested that possible safety issues of Cr2O3 NPs should be addressed promptly with limited future use in occupational settings.

Activation of Shc1 Allows Oncostatin M to Induce RANKL and Osteoclast Formation More Effectively Than Leukemia Inhibitory Factor.

Background and Purpose: The gp130 family of cytokines signals through receptors dimerizing with the gp130 subunit. Downstream signaling typically activates STAT3 but also SHP2/Ras/MAPK pathways. Oncostatin M (OSM) is a unique cytokine in this family since the receptor (OSMR) activates a non-redundant signaling pathway by recruitment of the adapter Shc1. We have studied the functional relevance of Shc1 for OSM-induced bone resorption.Experimental Approach: Osteoblasts were stimulated with OSM and STAT3 and Shc1 activations were studied using real-time PCR and Western blots. The role of STAT3 and Shc1 for OSM-induced RANKL expression and osteoclast formation was studied by silencing their mRNA expressions. Effects of OSM were compared to those of the closely related cytokine leukemia inhibitory factor (LIF).Key Results: OSM, but not LIF, induced the mRNA and protein expression of Shc1 and activated phosphorylation of Shc1 in the osteoblasts. Silencing of Shc1 decreased OSM-induced activation of STAT3 and RANKL expression. Silencing of STAT3 had no effect on activation of Shc1, but prevented the OSM-mediated increase of RANKL expression. Silencing of either Shc1 or STAT3 in osteoblasts decreased formation of osteoclasts in OSM-stimulated co-cultures of osteoblasts and macrophages. In agreement with these observations, OSM was a more potent and robust stimulator than LIF of RANKL formation and bone resorption in mouse calvariae and osteoclast formation in bone marrow cultures.Conclusions and Implications: Activation of the Shc1-dependent STAT3 signaling is crucial for OSM-induced osteoclast formation. Inhibition of Shc1 is a potential mechanism to specifically inhibit OSM-induced bone resorption.

Diagnostic issue on spontaneous osteonecrosis of medial tibial plateau.

To the Editor: I read with great interest the recent report of Yang et al[1], “Clinical Characteristics and Treatment of Spontaneous Osteonecrosis of Medial Tibial Plateau: A retrospective case study” (published on November 5, 2018, Chin Med J, page 2544–2550), because there have been very few reports on isolated spontaneous medial tibial plateau osteonecrosis and little is known about osteonecrosis of the tibial plateau. I would like to comment on diagnostic issues in this letter. Osteonecrosis of the knee can be a devastating disease that leads to end-stage arthritis of the knee. The knee is the second most commonly affected site after the hip.[2] Spontaneous osteonecrosis of the knee (SPONK) involving medial femoral condyle was described as a distinct entity by Ahlback et al in 1968[3] and is also called idiopathic or primary osteonecrosis to distinguish it from secondary osteonecrosis, which is associated with corticosteroid therapy, blood dyscrasias, Gaucher disease, caisson disease,[4] and other rare conditions, for example, laser-assisted arthroscopic chondroplasty.[5] The condition usually involves a single condyle, most often the medial femoral condyle, affected in 94% of the cases and can also occur in the lateral femoral condyle or in the tibial plateaus.[6] Involvement of the tibial plateau, which was first reported in the French literature by d’Angelijan et al[7] in 1967 and was described in the English literature by Houpt et al[8] is less common. Only 2% of osteonecrosis around the knee may affect the tibial plateau. The medial tibial plateau is more frequently affected than the lateral.[9] Therefore, to our knowledge, it remains a rare cause of knee pain. In the article, the case group contains 22 patients in 15 months (from March 2015 to June 2016), it means real prevalence may be underestimated. Magnetic resonance imaging (MRI) is both sensitive and specific for recognizing SPONK of both medial femoral condyle and tibial plateau and recommended for detection of the disease, due to its high sensitivity in detecting bone marrow edema.[10] MRI characteristics include a diffuse area of hyperintensity widespread into the metaphysics on T2-weighted images, the focal subchondral area of low signal intensity adjacent to the subchondral bone plate on T1-weighted images and focal epiphyseal contour depressions. But the MRI-detected subchondral bone marrow lesion, comprised of fibrosis, necrosis, edema, and bleeding into fatty marrow in different proportions as well as abnormal trabeculae, is also a common finding in patients with OA.[11] In the article, the MRI images showed in Figures 2 and 5 have no typical MRI signs of spontaneous medial tibial plateau osteonecrosis, MRI T1 and T2 images show cartilage degeneration, narrow of joint space, focal bone marrow edema and formation of subchondral cysts in medial knee compartment. We think it is more likely anteromedial osteoarthritis of knee, not a typical SPONK of medial tibial plateau. Although medial unicompartmental knee arthroplasty is also appropriate surgical indication. We also noted that in the series, there are 17 patients (77%) with a Level III medial meniscus posterior root tears (MMPRT) and the lesions of 68% patients involved the central tibial plateau. Subchondral marrow edema deep to the MMPRT was described as a harbinger of meniscal root failure. Ipsilateral tibiofemoral compartment bone marrow edema and insufficiency fractures are commonly noted in the presence of posterior meniscal tears.[12] Therefore, differential diagnosis is important and the condition has not been clearly discussed. Funding This work was supported by a grant from Ministry of Science and Technology of China (No. 2017YFC0108003). Conflicts of interest None.

DNA polymerase kappa counteracts inflammation-induced mutagenesis in multiple organs of mice.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

Effect of grape seed extract on maternal toxicity and in utero development in mice treated with zearalenone.

The aims of this study were to determine the polyphone content of grape seed extract (GSE) and to assess their protective effects against zearalenone (ZEN)-induced maternal toxicity and in utero development defects in mice. Five groups of pregnant mice were treated orally during days 6-13 of gestation as follows: control group, corn oil as vehicle (0.1ml/mice)-treated group, ZEN-treated group (25mg/kg b.w), GSE-treated group (150mg/kg b.w.), and ZEN plus GSE-treated group. All animals were sacrificed on the 19th day of gestation and samples of bone marrow were collected for the micronucleus assay. The maternal and developmental toxicity were carried out. The HPLC analyses revealed that GES is rich in gallic acid, syringic acid, vanillin, quercetin, and coumaric acid. ZEN administration resulted in severe maternal and developmental toxicity which included an increase of micronuclei formation in bone marrow, decreased maternal weight gain, and litter weight. It also induces fetal growth retardation, increased number of the aborted dams and resorbed fetuses, abnormality of fetal bone ossification, and number of fetuses with a hematoma. GSE showed positive effects on the pregnant mice and the developing fetuses. Moreover, it counteracted the detrimental effects of ZEN in dams and fetuses. It could be concluded that polyphenols in GSE are a promising candidate to protect against ZEN toxicity in highly endemic areas.

Bone marrow-derived mesenchymal stem cells enhance bone marrow regeneration in dental extraction sockets.

Bone marrow-derived mesenchymal stem cells (BMMSCs) remain the most widely used source of osteogenic cells in bone tissue engineering research. A cell-based treatment for alveolar ridge augmentation has received attention as an alternative to bone grafting. In the present study, BMMSC transplantation into tooth extraction sockets of C57BL/6J mice was evaluated for alveolar ridge regeneration. The first right maxillary molars were extracted, and then BMMSCs (PDGFRα+ Sca-1+ CD45- TER119- cells) isolated from femoral and tibial bone marrow were immediately transplanted into the extraction sockets. A control group underwent the same procedure except for BMMSC transplantation. Bone formation in the sockets was evaluated using micro-computed tomography and histological and immunohistochemical analyses. At 3 weeks, bone formation in the sockets was more advanced in the experimental group than in the control group. Histological analysis at 6 weeks after transplantation showed that the sockets in the experimental group also contained a greater quantity of bone marrow. Interestingly, socket bone mineral density was lower in the experimental group than in the control group at 6 weeks. These findings suggest that BMMSC transplantation accelerates bone healing and augments bone marrow formation in tooth extraction sockets.

Protection Against Radiation-Induced Micronuclei in Rat Bone Marrow Erythrocytes by Curcumin and Selenium L-Methionine.

The search for potent radioprotective agents for the amelioration of radiation side effect is an important aim in radiobiology. The present study aimed to evaluate the effects of curcumin and seleno-L-methionine against radiation-induced micronucleus formation in rat bone marrow. In total, 40 male rats were divided into 8 groups (n=5 each), including control, curcumin or seleno-L-methionine treated alone or in combination, 2 Gy irradiation, irradiation of treated groups with curcumin or seleno-L-methionine or their combination. Curcumin was administrated orally and seleno-L-methionine was injected intraperitoneally 24 hours before irradiation. The frequency of micronucleated normochromatic erythrocytes (MnNCEs) and micronucleated polychromatic erythrocytes (MnPCEs) was scored in 5,000 polychromatic erythrocytes (PCEs) and the cell proliferation ratio [(PCE/(PCE+NCE); NCE=normochromatic erythrocytes] was calculated for each treatment group. Data were analyzed by the SPSS software version 16.0 and P<0.05 was considered as statistically significant differences. Pretreatment with curcumin and seleno-L-methionine before irradiation reduced the frequency of MnPCEs and MnNCEs (P=0.01) and increased the cell proliferation ratio. Moreover, the results showed that this pretreatment reduced the frequency of MnPCEs with a protection factor (PF) of 1.2 and 1.6, respectively. The combination of curcumin and seleno-L-methionine in reducing MnPCEs and MnNCEs was not more effective than each agent alone, while improved cell proliferation ratio. Both curcumin and seleno-L-methionine showed potent protection against radiation induced MN in bone marrow cells. The combination of the two agents further ameliorates this activity, thus leading to improve bone marrow protection.

The results of experimental lumbar posterior-lateral fusion with platelet rich fibrin

In the case of instrumental spine fusion with the achieved biomechanical rigidity of the operated segment, the use of platelet-rich fibrin to stimulate osteogenesis is promising.Objective: to study the possibility of lumbar spine fusion under conditions of platelet-rich fibrin usage in the experiment.Methods: fusion modeling was made in 42 mature male Californian rabbits, which were divided into 6 groups of 7 individuals in each group In the 1st group we did not use grafts (control group); in the 2nd group we used bone autografts; in the 3rd — bone autografts with platelet-rich fibrin; in the 4th — allografts from iliac crest; in the 5th — a llografts f rom i liac c rest c ombined with platelet-rich fibrin; in the 6th — platelet-rich fibrin. Methods: roentgenological, morphological, statistical.Results: false positive fusion was revealed on X-rays in the 1st group in 28.6 % of cases, in the group 6th — in 7.1 %, false negative — in the 2nd and t he 3rd groups — equal in 7.1 %. In the control group 1st spine fusion was not found. In all other groups we have found the callous formation with different amount of bone, cartilage and connective tissue. In these groups on radiographs, the optical density of the newly formed bone exceeded the same parameters of the cancellous bone of the adjacent vertebral bodies.Conclusions: the greatest osteogenic properties were found in the case of a combination of bone autografts with platelet-rich fibrin: the highest incidence of fusion (71.4 %) was recorded, in which the predominantly mature bone tissue was formed. Regardless of the type of the graft (auto- or allo-), platelet-rich fibrin increased its reparative capabilities, providing a higher frequency of bone marrow formation based on the results of radiography a nd h istology.

The impact of collagen sponge composite bone marrow mesenchymal stem cells (BMSCs) in inducing interbody fusion.

To assess the effectiveness of bone mesenchymal stem cells (BMSCs) in the induction of interbody fusion. The 3rd generation BMSCs were seeded on collagen sponge scaffold and cultured in osteoblast induction medium for 3 weeks to prepare cell-scaffold complex. Thirty patients were randomly divided into three groups to establish the L4/L5 interbody fusion model. The cell-scaffold complex was implanted in the intervertebral space in group I, the collagen sponge scaffold was implanted in group II, and the autologous iliac crest spongy bone was implanted in group III. Palpation, radiography, micro-CT, and histology were performed on the 12th weeks after operation to evaluate osteogenesis and spinal fusion. BMSCs differentiated into osteoblasts in the cell-scaffold complex after osteogenic induction for 3 weeks. The spinal fusion rates in group I, II, and III were 40%, 0%, and 70%, respectively. Micro-CT and histological examination showed mature bone marrow and trabecular bone formation in the fusion segments. The new bone was integrated with the upper and lower vertebral body. The bone trabecula in group III was stronger than group I. The surgical segments in group II was scar tissue without collagen sponge residue. BMSCs can induce osseous fusion in the lumbar vertebra.

Effects of Honey and Hydroxyapatite on Bone Healing in Rats

Background: Bone defect healing in orthopedic surgery is a huge challenge. Nowadays, scaffolds are frequently used for tissue regeneration and engineering. One of these scaffolds, hydroxyapatite, is the most important inorganic material in bone, and a good option for promoting healing of bone defects. Honey has long been used for various wound healing with beneficial outcomes in the healing process. However, limited studies have addressed its use in repairing hard tissue lesions. Objectives: The aim of this study was to evaluate the effects of honey and hydroxyapatite on the restoration of bone defect in rats. Methods: In this study, 15 adult male rats weighing 200 to 300 grams were used. Rats were randomly assigned to three groups. A segment of the radius bone twice the size of the radius bone width was removed. In the first group (N = 5), the induced defect in the right hand was replaced with autograft bone using the same piece of bone, while the left hand remained empty without any implantation. In the second group (N = 5), defect in the right hand was filled with 0.5 g of honey and in the left hand with honey and autograft bone. In the third group (N = 5), defects in the right hand were filled with granules of hydroxyapatite and those in the left hand with 0.5 g combination of granules and honey. Radiographs in rats were taken at weeks two, four, six, and eight after surgery and bone biopsy for histopathological study was performed at week eight. Results: Radiographs and tissue samples were statistically analyzed in terms of bone formation, union and remodeling. Analyses showed that in the second week, honey-autograft was significantly better than the other groups (P < 0.05). The honey-alone treated group at all times was weaker than others. Histopathological investigations revealed that the hydroxyapatite-alone group was significantly different from hydroxyapatite-honey, autograft, honey, and negative control counterparts in terms of bone marrow formation (P < 0.05). Conclusions: Overall, this study showed that honey and hydroxyapatite alone is not effective in repairing bone defect yet in combination they function more effectively.

Achyranthes bidentata polysaccharide suppresses osteoclastogenesis and bone resorption via inhibiting RANKL signaling.

Osteoclasts are highly differentiated multinucleated giant cells that play fundamental roles in bone resorption and in the pathogenesis of osteolytic conditions, such as osteoporosis and cancer-induced bone loss. Achyranthes bidentata polysaccharide (ABP) is a hydrophilic compound with anti-oxidation and anti-aging characteristics. The impact of ABP on RANKL-induced osteoclast formation and bone resorption has not been assessed, hence, in this study we investigated the effect of ABP on osteoclast formation and resorption in murine bone marrow derived osteoclasts. We found that ABP was able to suppress RANKL-induced osteoclast differentiation and bone resorption activity at concentrations above 6.5 µM, while demonstrating no cytotoxicity at concentrations up to 10 µM. The actions of ABP were mediated through inhibition of RANKL-induced c-Fos and NFATc1 gene and protein expression. Furthermore, we found that ABP suppressed NFATc1 transcriptional activity, and the phosphorylation of MAPK pathways induced by RANKL. Collectively, ABP attenuates RANKL-mediated osteoclast activity and signaling, and might serve as a potential therapeutic candidate for preventing bone loss related diseases.

The periosteum-like effect of fresh human amniotic membrane on bone regeneration in a rabbit critical-sized defect model.

The purpose of this study was to investigate the effect of fresh human amniotic membrane (FHAM) as a substitute of periosteum to enhance bone regeneration in critical-sized defects. Tibial diaphyseal bone defects were created in forty New Zealand white rabbits and treated with FHAM or left empty. Treatment groups consisted of: FHAM implanted in the place of removed periosteum (FHAMP group); FHFAM implanted to fill the entire defect (FHAMF group) compared to negative control group; empty defect with removing the periosteum (NC group) and positive control group; and empty defect without removing the periosteum (PC group). Bone regeneration was evaluated by radiographic, micro-computed tomography (μ-CT) and histological analyses at 4 and 8weeks post-surgery. Radiographic and μ-CT analysis demonstrated clearly enhanced new bone formation in positive control group (PC) and FHAMP group compared to negative control group (NC) and FHAMF group. Histological staining exhibited remaining woven bones and cartilage matrix in the FHAMP group, immature lamellar bone with medellury cavity and marrow bone formation in PC group from 4weeks post-operatively. For FHAMF group, a little new bone formation was detected only from 8weeks post-operatively, and an absence of any sign of healing in NC group at both time points. The results provide that FHAM increases bone regeneration in critical-sized defects when it is implanted in the place of the removed periosteum, but its additive effect does not have the same effect of the natural periosteum.