Intra-bone marrow injection with engineered Lactococcus lactis for the treatment of metastatic tumors: Primary report.

Bone marrow has the capacity to produce different types of immune cells, such as natural killer cells, macrophages, dendritic cells (DCs) and T cells. Improving the activation of immune cells in the bone marrow can enhance the therapy of bone metastases. Previously, we designed an engineered probiotic Lactococcus lactis, capable of expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand (FOLactis), and proved that it can induce the activation and differentiation of several immune cells. In this research, we successfully establish mouse models of bone metastasis, lung metastasis and intraperitoneal dissemination, and we are the first to directly inject the probiotics into the bone marrow to inhibit tumor growth. We observe that injecting FOLactis into the bone marrow of mice can better regulate the immune microenvironment of tumor-bearing mice, resulting in a tumor-suppressive effect. Compared to subcutaneous (s.c.) injection, intra-bone marrow (IBM) injection is more effective in increasing mature DCs and CD8+ T cells and prolonging the survival of tumor-bearing mice. Our results confirm that IBM injection of FOLactis reprograms the immune microenvironment of bone marrow and has remarkable effectiveness in various metastatic tumor models.

Intra-bone marrow injection of magnesium isoglyrrhizinate inhibits inflammation and delays osteoarthritis progression through the NF-κB pathway.

OBJECTIVE: Osteoarthritis (OA) presents cartilage damage in addition to chronic inflammation. However, self-recovery of damaged cartilage in an inflammatory environment is not possible. Mesenchymal stem cells (MSCs) in the bone marrow are a source of regenerative repair of damaged cartilage. To date, whether intra-luminal administration of the bone marrow can delay the progression of OA is still unknown. This study, therefore, aimed to explore the role of intra-bone marrow injection of Magnesium isoglycyrrhizinate (MgIG) in delaying the OA progression and to investigate the underlying mechanism. METHODS: Rabbit OA models were established using the anterior cruciate ligament transection method while a catheter was implanted into the bone marrow cavity. 1 week after surgery, MgIG treatment was started once a week for 4 weeks. The cartilage degradation was analyzed using hematoxylin-eosin staining, Masson's trichrome staining and Alcian blue staining. Additionally, the pro-inflammatory factors and cartilage regeneration genes involved in the cartilage degeneration and the underlying mechanisms in OA were detected using enzyme-linked immunosorbent assay, quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS: The results of histological staining revealed that intra-bone marrow injection of MgIG reduced degeneration and erosion of articular cartilage, substantially reducing the Osteoarthritis Research Society International scores. Furthermore, the productions of inflammatory cytokines in the bone marrow cavity and articular cavity such as interleukin-1ß(IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) were inhibited upon the treatment of MgIG. At the same time, the expression of alkaline phosphate, tartrate-resistant acid phosphatase-5b (TRAP-5b) and C-telopeptides of type II collagen (CTX-II) in the blood also decreased and was positively correlated. On the contrary, cartilage-related genes in the bone marrow cavity such as type II collagen (Col II), Aggrecan (AGN), and SRY-box 9 (SOX9) were up-regulated, while matrix metalloproteinase-3 (MMP-3) was down-regulated. Mechanistically, MgIG was found to exert an anti-inflammatory effect and impart protection to the cartilage by inhibiting the NF-κB pathway. CONCLUSION: Intra-bone marrow injection of MgIG might inhibit the activation of the NF-κB pathway in the progression of OA to exert an anti-inflammatory effect in the bone marrow cavity and articular cavity, thereby promoting cartilage regeneration of MSCs in the bone marrow, making it a potential new therapeutic intervention for the treatment of OA.

Intra-iliac bone marrow injection as a novel alternative to intra-tibial inoculation in rat model.

BACKGROUND: Intra-bone marrow injection (IBMI) in rats is adopted in many studies for stem cell and hematopoietic cell transplantation. IBMI in the tibia or the femur results in severe distress to the animal. Therefore, this study aims to evaluate intra-iliac injections as an alternative approach for IBMI. METHODS: Twenty-seven Sprague Dawley rats were assigned into 3 groups, 9 rats each, for 4 weeks. The control group rats were not injected. Tibia group rats were injected intra-tibial and the iliac group rats were injected intra-iliac with saline. Behavioral, radiological, histopathological, and stress evaluation was performed. Total bilirubin, cortisol, and insulin-like growth factor-1 (IGF1) were measured. RESULTS: Behavioral measurements revealed deviation compared to control, in both injected groups, on the 1st and 2nd week. By the 3rd week, it was equivalent to control in the iliac group only. Bilirubin and cortisol levels were increased by intra-tibial injection compared to intra-iliac injection. The IGF-1 gene expression increased compared to control at 1st and 2nd weeks in intra-iliac injection and decreased by intra-tibial injection at 2nd week. The thickness of the iliac crest was not different from the control group, whereas there were significant differences between the control and tibia groups. Healing of the iliac crest was faster compared to the tibia. In the 3rd week, the tibia showed fibrosis at the site of injection whereas the iliac crest showed complete bone reconstruction. CONCLUSION: Intra-iliac injections exert less distress on animals, and by 3 weeks, they regained their normal activity in comparison to intra-tibial injections.

Cotransplantation of Umbilical Cord Mesenchymal Stem Cells Promotes the Engraftment of Umbilical Cord Blood Stem Cells in Iron Overload NOD/SCID Mice.

Iron overload aggravates the difficulty of umbilical cord blood (UCB) stem cell engraftment and reduces the survival of patients undergoing hematopoietic stem cell (HSC) transplantation. Mesenchymal stem cells (MSCs) have been suggested to have a significant role in HSC engraftment. This study aimed to determine the effect of intra-bone marrow (IBM) and i.v. cotransplantation of UBC mononuclear cells (MNCs) and umbilical cord (UC) MSCs on engraftment and hematopoietic recovery in an iron overload hematopoietic microenvironment. The iron overload model was established by dose-escalation intraperitoneal injection of iron dextran in NOD/SCID mice. Iron deposition in the bone marrow, heart, and liver was examined using hematoxylin and eosin (H&E) staining. Serum levels of ferritin and iron status in the liver were measured. The iron overload NOD/SCID mice were sublethally irradiated and divided into 5 groups for transplantation: (1) control group; (2) IBM+ group: IBM injection of combined UCB-MNCs/UC-MSCs; (3) IV+ group: i.v. injection of combined UCB-MNCs/UC-MSCs; (4) IBM group: IBM injection of only UCB-MNCs; and (5) IV group: i.v. injection of UCB-MNCs. At 6 weeks after transplantation, the human CD45+ cells in the bone marrow were analyzed by flow cytometry. The semiquantitative analysis of vascular endothelial growth factor (VEGF-A), osteopontin (OPN), and stromal cell-derived factor-1a (SDF-1a) were examined by immunohistochemical staining (IHC). Compared with the IBM and IV groups, the survival rate and the percentages of human CD45+ cells and CD34+ cells and colony-forming units (CFU) in bone marrow were elevated in the IBM+ and IV+ groups. In addition, the levels of VEGF-A, OPN, and SDF-1a in bone marrow were all higher in the IBM+ and IV+ groups. Our data show that IBM and i.v. cotransplantation of UC-MSCs can improve the engraftment and proliferation of UCB-MNCs in iron overload NOD/SCID mice. The increased expression of VEGF-A, OPN, and SDF-1a in the bone marrow may be involved in improving the hematopoietic microenvironment and promoting the implantation of human UCB stem cells in the bone marrow with iron overload.

Intra-bone marrow injection of fetal liver mononuclear cells to reconstruct the hematopoietic system in mice / 上海交通大学学报（医学版）

Objective:To observe the effect of intra-bone marrow injection (IBMI) of fetal liver mononuclear cell suspension on reconstructing the hematopoietic system in mice. Methods : The fetal liver mononuclear cells from the mice with gestational age of 13.5 days were obtained by Ficoll separation technique, and then the hematopoietic stem cells (HSCs) were obtained by magnetic bead sorting. The fetal liver mononuclear cells were injected into the tibias of 137Cs irradiated mice by IBMI. The recipient mice were observed for reconstitution of the hematopoietic system by routine blood examination, blood smear and bone marrow smear. The donor HSCs in the bone marrow of the recipient mice was subjected to flow cytometry 1 year after transplantation. Results: HSCs accounted for 0.171% in the mouse fetal liver mononuclear cells. One week after transplantation of mouse fetal liver mononuclear cells, blood cells derived from donor cells were detected from peripheral blood; in the third week after transplantation, peripheral blood leukocytes, red blood cells and hemoglobin of recipient mice were restored to the pre-irradiation levels; the percentage of donor-derived blood cells in peripheral blood in the fifth week was stable; the HSCs in the bone marrow of recipient mice were derived from donor cells 1 year after transplantation. Conclusion: The murine fetal liver mononuclear cells can efficiently reconstruct the hematopoietic system of mice by IBMI.

CD34-negative hematopoietic stem cells show distinct expression profiles of homing molecules that limit engraftment in mice and sheep.

We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34-) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin- or 18Lin-) CD34+/- cells from human CB in mice and sheep. Both 13Lin- and 18Lin- CD34+ cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin- nor 18Lin- CD34- cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin- and 18Lin- CD34- cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34- HSCs is significantly more limited than that of CD34+ HSCs. As for 18Lin-, CD34- HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34- HSCs could thus explain in part their reduced ability to home to the BM niche.

Salvage technique of cryopreserved umbilical cord blood and nursing experience / 中国实用护理杂志

Objective To investigate the safety of infusion and effective care of cryopreserved umbilical cord blood. Methods Side effects happened during transfusion of cord blood in 78 patients of 111times of transfusion were observed and nursed. Results During intravenous transfusion way:1 case with acute hemolytic reaction; 1 case with sudden loss of consciousness and convulsions; 4 cases with severe headache, difficulty with breathing, rapid increase of blood; 46 patients with high blood pressure, heart rate or blood pressure, heart rate, 1 patient had symptoms of frequent premature beats; most of the cases have hemoglobinuria after the infusion. Transfusion of bone marrow injection of ways: 11 patients have radioactive soreness in leg, hemoglobinuria hemoglobinuria mild symptoms or no symptoms during the process of infusion. Conclusions Systemic reactions were the main side effects during intravenous transfusion of cord blood, conditions change fast and complex, Nursing should focus on preventive care and preparation,close observation and active and effective symptomatic treatment; reaction of bone marrow injection of umbilical cord blood was local pain, the focus of nursing was aseptic operation and injection time.

Effects of allogenic intra-bone marrow bone marrow transplantation on the hematopoiesis in mice / 基础医学与临床

Objective To investigate the effects of allogenic intra-bone marrow bone marrow transplantation (IBM-BMT) on re-establishing hematopoiesis in mice. Methods Bone marrow mononuclear cells (BMNCs) from BALB/ c mice were transplanted into the C57BL/6 mice treated with a lethal dose of ~(60)Coγ-ray radiation through intra-bone marrow injection or intravenous injection. Sixty of the C57BL/6 mice were randomly divided into three groups as higher dose intra-bone marrow injection group (IBM1 group), lower dose intra-bone marrow injection group (IBM2 group) and intravenous injection group (IV group). The nucleated cell numbers of whole bone marrow from the tibia of each recipient mouse were counted respectively at the day 1, day 3, day 6 and day 9 after the transplantation. The donor-derived total nucleated cells and myeloid cells were quantified by flow cytometry. Results At 6th day after transplantation, more total bone marrow nucleated cells, total donor-derived nucleated cells and donor-derived myeloid cells in the tibia of injected side in both IBM1 group and IBM2 group were found than that in IV group (P<0.05 or P<0.01). Conclusion Compared with traditional bone marrow transplantation (IV-BMT),IBM-BMT improves the bone marrow hematopoiesis in the early hematopoietic re-establishing stage in allogenic bone marrow transplantation.