BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells.

Bone regeneration achieved using mesenchymal stem cells (MSCs) and nonviral gene therapy holds great promise for patients with fractures seemingly unable to heal. Previously, MSCs overexpressing bone morphogenetic proteins (BMPs) were shown to differentiate into the osteogenic lineage and induce bone formation. In the present study, we evaluated the potential of osteogenic differentiation in porcine adipose tissue- and bone marrow-derived MSCs (ASCs and BMSCs, respectively) in vitro and in vivo when induced by nucleofection with rhBMP-2 or rhBMP-6. Our assessment of the in vivo efficiency of this procedure was made using quantitative micro-computed tomography (micro-CT). Nucleofection efficiency and cell viability were similar in both cell types; however, the micro-CT analyses demonstrated that in both ASCs and BMSCs, nucleofection with rhBMP-6 generated bone tissue faster and of higher volumes than nucleofection with rhBMP-2. RhBMP-6 induced more efficient osteogenic differentiation in vitro in BMSCs, and in fact, greater osteogenic potential was identified in BMSCs both in vitro and in vivo than in ASCs. On the basis of our findings, we conclude that BMSCs nucleofected with rhBMP-6 are superior at inducing bone formation in vivo than all other groups studied.

Ovariectomy and genes encoding core circadian regulatory proteins in murine bone.

SUMMARY: This study investigated the influence of ovarian hormone deficiency on core circadian regulatory protein (CCRP) in the context of bone loss. Our data suggest that ovarian hormone deficiency disrupts diurnal rhythmicity and CCRP expression in bone. Further studies should determine if chronobiology provides a novel therapeutic target for osteoporosis intervention. INTRODUCTION: CCRP synchronize metabolic activities and display an oscillatory expression profile in murine bone. In vitro studies using bone marrow mesenchymal stromal/stem cells have demonstrated that the CCRP is present and can be regulated within osteoblast progenitors. In vivo studies have shown that the CCRP regulates bone mass via leptin/neuroendocrine pathways. The current study used an ovariectomized murine model to test the hypothesis that ovarian hormone deficiency is associated with either an attenuation and/or temporal phase shift of the CCRP oscillatory expression in bone and that these changes are correlated with the onset of osteoporosis. METHODS: Sham-operated controls and ovariectomized female C57BL/6 mice were euthanized at 4-h intervals 2 weeks post-operatively. RESULTS: Ovariectomy attenuated the oscillatory expression of CCRP mRNAs in the femur and vertebra relative to the controls and reduced the wheel-running activity profile. CONCLUSION: Ovarian hormone deficiency modulates the expression profile of the CCRP with potential impact on bone marrow mesenchymal stem cell lineage commitment.

Ultrasound-based nonviral gene delivery induces bone formation in vivo.

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

Stem cells as vehicles for orthopedic gene therapy.

Adult stem cells reside in adult tissues and serve as the source for their specialized cells. In response to specific factors and signals, adult stem cells can differentiate and give rise to functional tissue specialized cells. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into various mesenchymal lineages such as muscle, bone, cartilage, fat, tendon and ligaments. Adult MSCs can be relatively easily isolated from different tissues such as bone marrow, fat and muscle. Adult MSCs are also easy to manipulate and expand in vitro. It is these properties of adult MSCs that have made them the focus of cell-mediated gene therapy for skeletal tissue regeneration. Adult MSCs engineered to express various factors not only deliver them in vivo, but also respond to these factors and differentiate into skeletal specialized cells. This allows them to actively participate in the tissue regeneration process. In this review, we examine the recent achievements and developments in stem-cell-based gene therapy approaches and their applications to bone, cartilage, tendon and ligament tissues that are the current focus of orthopedic medicine.

Synthesis and biodegradation of arabinogalactan sponges prepared by reductive amination.

The synthesis of polysaccharide-based sponges for the use in tissue engineering was systematically investigated. A comparison study of the branched polysaccharide arabinogalactan (AG) and the linear polysaccharide dextran in the formation of sponges by the reaction with diamines or polyamines was conducted. Three AG-based sponges were synthesized from the crosslinking reaction with different amine molecules. The sponges obtained were highly porous, rapidly swelled in water, and were stable in vitro for at least 11 weeks in aqueous media at 37 degrees C. AG-chitosan sponges were chosen as most suitable to serve as scaffolds for cell growth in tissue engineering. The biocompatibility in vivo of these sponges was evaluated by histological staining and non-invasive MRI technique after implantation in BALB/c mice. The sponge evoked an inflammatory response with vascularization of the implant. The inflammatory reaction decreased with time, indicating a healing process.

Mesenchymal stem cells for bone gene therapy and tissue engineering.

Mesenchymal Stem Cells (MSCs) are adult stem cells that constitute a variety of adult tissues. MSCs maintain self-renewal ability with the ability to give rise to different mesenchymal cells, and are therefore responsible in part, for the regenerative capacity of mesenchymal tissues. MSCs throughout a variety of species were found to be able to differentiate to several mesenchymal tissues including: bone, cartilage, stroma, adipose, connective tissue, muscle and tendon. MSCs are relatively easily isolated from the bone marrow and expanded in vitro. It was found that MSCs play an important role in bone physiology and hematopoiesis, and in part participate in the pathophysiology related to bone diseases, mainly osteoporosis. MSCs were widely used in experimental studies in vivo, and were shown to form mesenchymal tissues. These discovered features have made MSCs good candidates for the development of various therapeutic modalities aimed to regenerate mesenchymal tissues, mainly bone. The more important approaches currently utilizing MSCs are gene therapy and tissue engineering. Both exploit the current knowledge in molecular biology and biomaterial science in order to direct MSCs to differentiate in vivo to desired lineages and tissues. Better understanding of the molecular mechanism directing the differentiation of MSCs, will eventually allow us to properly manipulate MSCs both in vivo and ex vivo to allow the regeneration of complex tissues and organs.

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice.

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100-120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1-15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5 x 10(4) Cetus units twice daily for 4-5 days), 5-FU augmented (up to 37-fold, days 1-9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3-4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3-9), as determined by limiting-dilution cultures with IL-2 (for 7-8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6-9 days after treatment with 5-FU, Adriamycin or decarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6-12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1- LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.

Chemo-immunotherapy in patients with metastatic melanoma using sequential treatment with dacarbazine and recombinant human interleukin-2: evaluation of hematologic and immunologic parameters and correlation with clinical response.

We have treated 18 patients with metastatic malignant melanoma (MM) with high-dose IL-2 administered by continuous iv infusion in combination with dacarbazine (DTIC), and correlated the clinical response with various hematologic and immunologic parameters. Two regimens differing in the sequence of treatment were employed, and 1-6 treatment cycles were given, depending on patient response. Two patients had a complete response (CR, 46+m, 14m), two patients a partial response (PR, 16m,6m), one a minimal response and four had a stable disease lasting 2-7 months, thus the response rate (CR+PR) was 22%. None of the following parameters, tested prior to initiation of the therapy and 1-2 days after termination of each course of IL-2, correlated with the clinical response: WBC counts (total and differential), levels of blood CD4 and CD8 T cells, NK cells, monocytes and B cells, production of IL-1 and IL-1 inhibitor by monocytes, responsiveness to 3 mitogens, NK/LAK cell activity, and serum levels of IL-1 alpha, IL-2, soluble IL-2 receptor, and TNF alpha. The only prognostic parameter was the greater increase in the level of IL-2 receptor (Tac)-bearing lymphocytes in the responding patients after 1-3 cycles of IL-2. The data suggests that non-specific immune parameters have no prognostic value for patients undergoing IL-2-based immunotherapy.

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines.

The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon alpha A/D (IFN alpha), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6-18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN alpha were administered three to five times weekly for 1-3 weeks, usually starting 2-5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN alpha, IL-2 + IFN alpha +/- LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN alpha. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN alpha + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN alpha. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.