Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris.

Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates.

Evaluation of different scaffolds for BMP-2 genetic orthopedic tissue engineering.

To better understand the effects of scaffold materials for bone morphogenetic protein 2 (BMP-2) genetic tissue engineering in vivo, several gels, including alginate, collagen, agarose, hyaluronate, fibrin, or Pluronic, were mixed with adenovirus-mediated human BMP-2 gene (Adv-hBMP-2) transduced bone marrow stromal cells (BMSCs) and injected into the muscles of athymic mice to evaluate the resulting osteogenesis and chondrogenesis. These gel and gene-transduced BMSC mixtures were also loaded onto beta-TCP/HAP biphasic calcined bone (BCB) and observed under scanning electron microscopy (SEM). In addition, these composite scaffolds were implanted into the subcutaneous site of athymic mice to construct tissue-engineered bone. After injection, collagen, hyaluronate, or alginate gel mixed with gene-transduced BMSCs induced more bone formation than a cell suspension in alpha-MEM. The agarose-gene-transduced BMSC gel was found to contain much more hyaline cartilage. SEM showed the BMSCs could survive in alginate, agarose, and collagen gel in vitro for up to 8 d. After implantation of tissue-engineered bone, the alginate, collagen, and agarose gel could promote new bone formation within a BCB in vivo. Little or no bone formed after injection of fibrin or Pluronic gel mixed with BMSCs or implantation with BCB. These findings help to elucidate the effects of various scaffold materials for future research in orthopedic tissue engineering using BMP-2 transduced cells.

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice.

OBJECTIVE: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). METHODS: Goat bone marrow-derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene (Group 1), Adv-beta gal transfected MSCs (Group 2) and uninfected MSCs (Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. RESULTS: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. CONCLUSIONS: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.

[Effects of bone morphogenetic protein-2 gene therapy on the bone-implant interface: an experimental study with dogs].

OBJECTIVE: To investigate the effects of bone morphogenetic protein-2 (BMP-2) gene therapy on the bone-implant interface in the reconstruction of periprosthetic bone defect. METHODS: Transverse defects were caused in the external condylae of both femurs of 14 adult Beagle dogs. Titanium alloy implants were inserted and a bone defect 3 mm wide around the titanium alloy implant was preserved. Then the total 28 defects were divided into 4 groups: 8 bone defects remained untreated (blank control group); 8 bone defects were implanted with heterogeneous freeze-dried bone by impaction grafting technique (non-cell group); 8 bone defects were implanted with heterogeneous freeze-dried bone loaded with autogenous bone marrow stromal cells (BMSCs) from the greater trochanter of the same dog (cell group); and 10 bone defects were implanted with freeze-dried allograft loaded with autogenous BMSCs from the greater trochanter of the same dog which were transfected by Adv-BMP-2 gene (gene group). Three, 6, and 12 weeks after implantation X-ray examination was carried out to observe the place of the implant and the absorption of the implants. Six and 12 weeks after the dogs were killed and their bone defects were taken out to undergo histological, histomorphometric and biomechanical examination to observe the healing and oseeointegration of the bone-implant interface. RESULTS: Histological examination showed that 6 weeks after implantation new bone formation was found on the implant surface and there was point contact between the bone and implant in the gene group with the bone-to-impact contact (BIC) of about 10%; and continuous soft tissue was found at bone-implant interface in all other groups. Twelve weeks after, there was thick soft tissue membrane between the new bone and implant in the blank control group; most of the interface was connective fibrous tissue in the non-cell group and cell group with point contact between the bone and implant and a BIC lower than 10%; and in the gene group the interface consisted mainly of bone tissue and continuous bone-implant contact was found with the BIC of 50%, significantly higher than those of the other 2 groups (both P < 0.01). The mechanical strength of interface increased time-dependently in all groups, that of the gene group being significantly higher than those of the other 2 groups at any time-points (both P < 0.01). CONCLUSION: BMP-2 gene therapy can improve the osseointegration of bone-implant interface.

MicroRNA 302/367 Cluster Effectively Facilitates Direct Reprogramming from Human Fibroblasts into Functional Neurons.

Recent studies suggest that mature somatic cells can be reprogrammed to become induced pluripotent stem cells by overexpressing specific transcription factors or microRNAs (miRNAs). Theoretically, this technique could provide a wide array of cells for therapeutics. However, the process of redifferentiation after cell reprogramming to pluripotency is inefficient and time restricted. We proposed that the differentiation of somatic cells into specific cells of another germ layer can be induced and accelerated with appropriate miRNAs and culture conditions. In human fibroblasts, we found that overexpression of pluripotency stem cell-specific miRNA-302/367 cluster, together with two other neuron-specific miRNAs (miRNA-9/9* and miRNA-124) induced fibroblasts conversion into neurons. The cells assumed neuron morphology, were positive for several neuron markers, and exhibited neuronal membrane potential feature. Moreover, concentrated expression of synaptic markers were observed in these cells in vitro and in vivo in nude mice brain, suggesting possible connectivity. To achieve efficient reprogramming, miRNA-302/367 cluster, miRNA-9/9*, and miRNA-124 were all required. The combination of the proved pluripotency-inducing miRNA-302/367 cluster and cell-specific miRNAs provides a unique strategy for one-step cellular conversion that could have important implications for studies of neuron development and neurological disease therapy.

Adenovirus mediated BMP-13 gene transfer induces chondrogenic differentiation of murine mesenchymal progenitor cells.

UNLABELLED: Chondrogenic/osteogenic differentiation of a mesenchymal progenitor stimulated by BMP-13 (CDMP-2) was studied. C3H10T1/2 cells were transduced by an adenoviral construct containing BMP-13 or BMP-2. BMP-13 supported chondrogenesis but not terminal differentiation, whereas BMP-2 stimulated endochondral ossification. The studies show that BMP-13 may fail to support terminal chondrocyte differentiation. INTRODUCTION: Bone morphogenetic protein (BMP)-13 is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors. Although the biological functions of BMP-13 remain poorly understood, continued postnatal expression of BMP-13 in articular cartilage suggests that this protein may function in an autocrine/paracrine fashion to regulate growth and maintenance of articular cartilage. The purpose of this study was to elucidate the role of BMP-13 in chondrogenic differentiation. MATERIALS AND METHODS: Replication-deficient adenoviruses carrying human BMP-13 (Adv-hBMP13), bacterial beta-galactosidase (Adv-beta gal), and human BMP-2 (Adv-hBMP2) were constructed. Murine mesenchymal progenitor cells (C3H10T1/2) were transduced with these vectors, and differentiation to the chondrogenic lineage was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), biochemical, and histological analyses. RESULTS AND CONCLUSIONS: Our findings revealed that hBMP-13 transduced cells differentiated into round cells that stained with Alcian blue. Analysis of gene expression in hBMP-13-transduced cells demonstrated presence of cartilage-specific markers, absence of hypertrophic chondrocyte specific markers, and upregulation of proteoglycan biosynthesis. In particular, hBMP-13-transduced cells had significantly less and delayed expression of alkaline phosphatase activity and calcium mineral accumulation than hBMP-2-transduced cells. Except for BMPR-IB/ALK-6, expression of BMP receptors was identified constitutively in C3H10T1/2 cells and was not affected by the presence of either of the BMPs. In summary, hBMP-13, while stimulating chondrogenesis, failed to support differentiation to hypertrophic chondrocytes and endochondral ossification similar to hBMP-2. Thus, this may prove to be a useful strategy for cell-based regeneration of articular cartilage.

Optimal treatment timing to attenuate neuronal apoptosis via Bcl-2 gene transfer in vitro and in vivo.

Although Bcl-2 gene transfer can rescue cells from neuronal apoptosis, the temporal relationship between treatment initiation time and effectiveness is unknown. The purpose of present study is to investigate the optimal treatment timing of Bcl-2 gene transfer in saving cells after neural insults. Bcl-2 gene transfer was mediated by recombinant adenovirus carrying human bcl-2 oncogene (Adv-Bcl-2). Adenovirus carrying beta-galactosidase gene (Adv-Bgal) served as a control. A serum withdrawal model of NSC-19 cell culture was used to induce apoptosis in vitro. At various time points before or after serum withdrawal, the motor neuron cells (NSC-19 cells) were infected with either Adv-Bcl-2 or Adv-Bgal. At 72 h after serum withdrawal, the number of apoptotic cells and DNA fragmentation were examined to evaluate the effect of Bcl-2 gene transfer. A weight-drop spinal cord injury model in rats was used as in vivo model. At various time points before or after experimental spinal injury, virus solution, including Adv-Bcl-2 or Adv-Bgal, was injected at the spinal cord in injured rats. The degree of cord injury was measured at 72 h after injury. TUNEL staining was performed to count cells that have undergone DNA damage in sections. Bcl-2 protein overexpression was confirmed by immunostaining both in vitro and in vivo model. In vitro, Adv-Bcl-2 infection produced a less prominent DNA laddering pattern. Adv-Bcl-2 infection between 24 h before and 4 h after serum withdrawal significantly reduced the apoptotic cell death. In vivo Adv-Bcl-2 injection immediately after injury effectively suppressed the injury lesion by blocking DNA fragmentation and irreversible cellular injury. Our data demonstrate that earlier initiation of Bcl-2 gene transfer can produce improved neural cell rescue following neural insults. These results stress important temporal considerations in future gene therapy strategies for spinal cord injury.

Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.

Bone marrow derived mesenchymal stem cells (MSC) have been shown to be progenitor cells for mesenchymal tissues. These cells may also provide a potential therapy for bone repair. Our previous studies showed that MSC engineered with the gene for bone morphogenetic protein 2 (BMP-2), a growth factor for bone cells, were capable of differentiating into osteoblast lineage and inducing autologous bone formation in several animal models. Culturing individual MSC for autologous implantation, however, remains problematic. The number of human MSC with osteogenic potential decreases with age, and, in certain diseases, the patient's marrow may be damaged or the healthy cells reduced in number. In this study, we used rats with a femoral segmental defect to investigate whether allogeneic BMP-2 engineered MSC would facilitate bone healing. We show that BMP-2 engineered allogeneic MSC can repair critical bone defects to the same degree as rats treated with BMP-2 engineered autologous MSC, if the allogeneic group receives short-term treatment with immunosuppressant FK506. We also show that allogeneic gene transferred MSC are directly involved in bone repair, in addition to acting as gene deliverers.

Thoracoscopic intradiscal spine fusion using a minimally invasive gene-therapy technique.

BACKGROUND: Gene therapy has been utilized to achieve posterior intertransverse process fusion in rodents. To our knowledge, however, no one has previously reported on the use of this technique to achieve anterior spinal fusion in mammals. The purpose of the present study was to determine if a gene-therapy technique can be utilized to achieve anterior intradiscal fusion in pigs with use of minimally invasive techniques. METHODS: Mesenchymal stem cells were isolated from each of three pigs, expanded in culture, and transduced with an adenovirus carrying either the gene for bone morphogenetic protein-2 (Adv-BMP2) or the control gene, beta-galactosidase (Adv-betagal). In vitro, assays were performed to detect BMP-2 expression as well as protein markers of bone formation. In vivo, four thoracic disc spaces in each of three pigs were injected thoracoscopically with cells after 1 cm (3) of the disc had been removed. In each of the three pigs, two discs were injected with autologous mesenchymal stem cells transduced with Adv-BMP2, the third disc was injected with cells transduced with Adv-betagal (control 1), and the fourth disc served as the sham-operated control (control 2). The three animals were killed six weeks after the implantation. Computerized tomographic scanning was performed on two of the specimens, and histological examination was performed on all specimens. The computerized tomographic scans and histological examinations were then interpreted in a blinded fashion. RESULTS: In the in vitro study, a human BMP-2 protein band was detected in the medium of Adv-BMP2-transduced stem cells but not in that of the control cells. The Adv-BMP2-transduced stem cells were associated with a fivefold increase in alkaline phosphatase activity compared with the controls as well as with matrix mineralization and increased protein expression of type-I collagen, osteopontin, and bone sialoprotein. In the in vivo study, radiographic examination demonstrated anterior spinal fusion in all six disc spaces that had been treated with implantation of Adv-BMP2-transduced stem cells. In contrast, the six control disc spaces had little or no intervening bone. Histological examination demonstrated bridging bone from end plate to end plate in all six disc spaces that had been treated with implantation of Adv-BMP2-transduced stem cells. The six control disc spaces had no bridging bone. CONCLUSION: The Adv-BMP2-transduced mesenchymal stem cells produced BMP-2 protein. Further, the cells differentiated into osteoblasts and induced anterior spinal fusion in six of six disc spaces in this pig model. Although many technical and practical challenges remain, the results of the present study suggest that it may eventually be possible to use similar techniques to achieve anterior spinal fusion in humans.

Ex vivo gene therapy in autologous critical-size craniofacial bone regeneration.

In therapeutic bone repairs, autologous bone grafts, conventional or vascularized allografts, and biocompatible artificial bone substitutes all have their shortcomings. The bone formed from peptides [recombinant human bone morphogenetic proteins (BMPs)], demineralized bone powder, or a combination of both is small in size. Tissue engineering may be an alternative for cranial bone repair. In this study, the authors developed an animal model to test the hypothesis that replication-defective, adenovirus-mediated human BMP-2 gene transfer to bone marrow stromal cells enhances the autologous bone formation for repairing a critical-size craniofacial defect. The mesenchymal stromal cells of miniature swine were separated from the iliac crest aspirate and expanded in monolayer culture 1 month before implantation. The cultured mesenchymal stromal cells were infected with recombinant, replication-defective human adenovirus BMP-2, 7 days before implantation. Bilateral 2 x 5-cm2 cranial defects were created, leaving no osteogenic periosteum and dura behind. Mesenchymal stromal cells at 5 x 10(7)/ml were mixed with collagen type I to form mesenchymal stromal cell/polymer constructs. Mesenchymal stromal cells used for the control site were infected with adenovirus beta-Gal under the same conditions. After 6 weeks and 3 months, 10 miniature swine were euthanized and the cranium repair was examined. Near-complete repair of the critical-size cranial defect by tissue-engineered mesenchymal stromal cell/collagen type I construct was observed. The new bone formation area (in square centimeters) measured by three-dimensional computed tomography demonstrated that the improvement from 6 weeks to 3 months was significantly greater on the experimental side than on the control side (2.15 cm2 versus 0.54 cm2, p < 0.001) and significantly greater at 3 months than at 6 weeks (2.13 cm2 versus 0.52 cm2, p < 0.001). The difference between the experimental and control groups was significant at 3 months (mean difference, 2.13 cm2; p < 0.001). The maximal compressive strength of the new bone was similar to that of the normal cranial bone when evaluated by biomechanical testing (cranium bone versus tissue-engineered bone, 88.646 +/- 5.121 MPa versus 80.536 +/- 19.302 MPa; p = 0.227). Adenovirus was absent from all constructs by immunochemical staining at 6 weeks and 3 months after implantation. The successful repair of cranial defects in this experiment demonstrates the efficacy of the integration of the autologous stem cell concept, gene medicine, and polymers in producing tissue-engineered bone.

[BMP-2 gene modified tissue-engineered bone repairing segmental tibial bone defects in goats].

OBJECTIVE: To evaluate the effectiveness of the tissue-engineered bone substitute loaded with adenovirus mediated human bone morphogenetic protein-2 gene (Adv-hBMP-2) transfected bone marrow derived mesenchymal stem cells (BMSC) in the repair of diaphyseal segmental bone defect of large animal. METHODS: The right tibial bone defects (2.6 cm) model of 26 goats were established and divided into 5 groups: I. Adv-hBMP-2 transfected BMSC/calcined bone (CB) group (n = 9); II. adenovirus-beta-galactosidase (Adv-betagal) gene transfected BMSC/CB group (n = 6); III. untransfected BMSC/CB group (n = 6); IV. single CB group (n = 3); VI. untreated group (n = 2). The above tissue-engineered bone substitutes were implanted in the bone defects respectively except group VI. Roentgenography, histomorphometrical analysis and biomechanical measurement were studied at various times. RESULTS: X-ray: at 4 - 8th weeks after implantation, more bony callus was found in the bone defects of group I. The complete healing rates of group I, II, III, IV, and V were 5/8, 1/5, 0/5, 0/2, 0/1 respectively at 26th week after implantation. Histomorphometrical analysis showed much more new bony callus including cortical bone formed in group I than those of other groups. The compression strength of the implanted bone substitute of group I is significantly higher than those of group II and III. CONCLUSION: The tissue-engineered bone substitute loaded with human BMP-2 gene transfected BMSC can repair diaphyseal segmental bone defect of large animal (goat).

[Preparation of a biphasic calcined bone of beta-TCP/HAP and its application in BMP-2 gene medicine].

In this study, we produced a biphasic absorbable calcined bone (CB) of beta-TCP/HAP, and evaluated its function as a carrier of bone marrow derived mesenchymal stem cells(BMSCs) in BMP-2 gene medicine. Biphasic CB was manufactured and its surface was coated by collagen. X-ray diffraction analysis, scanning electron microscopy and biomechanical measurement were performed on the product. Heterotopic bone induction of product as carrier of BMP-2 gene transferred BMSCs and its biodegradability were tested in nude mice and goats. X-ray diffraction analysis showed biphasic patterns of HAP and beta-TCP. Scanning electron microscopy showed the porosity were similar to those of the cancellous bone, and the adhesion of cells on the CB surface were better after surface-coating with collagen. It had certain biomechanical strength and appropriate biodegradability. Biphasic CB loaded with Adv-hBMP-2 transduced BMSCs could induce much bony callus at the subcutaneous site of nude mice and the tibial bone defects of goats. The results showed biphasic CB is a superior carrier of cells in BMP-2 gene medicine.

Reconstruction of peri-implant bone defects using impacted bone allograft and BMP-2 gene-modified bone marrow stromal cells.

Impaction bone allografting represents an attractive procedure for bone defects reconstruction in joint replacement. And it was found that bone morphogenetic protein-2(BMP-2) gene therapy can enhance bone healing. The purpose of this study was to determine if combined adenovirus mediated human BMP-2(Adv-hBMP-2) gene-modified bone marrow stromal cells(BMSCs) with allograft enhanced the defects healing and improved the strength of implant fixation in 3-mm bone defect around a titanium alloy implant. Using the impaction grafting technique, the defects were reconstructed using freeze-dried allograft, freeze-dried allografts loaded with autogenous BMSCs, or freeze-dried allografts loaded with autogenous BMSCs modified with the human bone morphogenetic protein-2 (hBMP-2) gene. At 6 and 12 weeks, the Bone-implant Contact rate and strength of the interface in the group with BMP-2 gene medication were significantly higher than those of the non-cell or cell groups. BMP-2 gene medication also showed significant effects on allograft healing and replacement compared with those of two other groups, as evidenced by increased new bone formation and reduced graft remnants. The results suggest that BMP-2 gene medication can enhance allograft healing and osseointegration of the bone-implant interface.

Immunomodulatory and osteogenic differentiation effects of mesenchymal stem cells by adenovirus-mediated coexpression of CTLA4Ig and BMP2.

Many reports have previously utilized a human bone morphogenetic protein 2 (BMP2)-expressing recombinant adenoviral vector (AdBMP2) and mesenchymal stem cells (MSCs) for osteoinductive gene therapy. However, immunosuppression is essential for osteoinduction by AdBMP2, and this is one of the major impediments to its clinical use. In the present study, in vitro propagated MSCs were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig). Lymphocyte response was induced by allogeneic-irradiated MSCs as stimulators. We also examined the effects of cotransfection with a combination of the CTLA4Ig and the BMP2 gene on osteoblastic cell differentiation. The results showed that BMP2 gene transfected MSC elicited significant stimulatory responses, and one-way MLR reactions were significantly blunted by CTLA4Ig. Further study demonstrates that cotransfection of MSCs with the combination of the CTLA4Ig and the BMP2 gene stimulates osteoblastic cell differentiation in vitro. The findings suggest that genetic engineering of MSCs to express an immunosuppressive molecule in combination with an osteogenic protein gene may have potential application in the treatment of several genetic diseases and in bone reconstruction.

[The effect of HA mixed with adenovirus mediated rhBMP-2 transferred BMSCs of goats on distraction osteogenesis].

OBJECTIVE: To evaluate the effectiveness of HA mixed with adenovirus mediated rhBMP-2 gene (Adv-rhBMP-2) transferred BMSCs of goats on distraction osteogenesis. METHODS: Nineteen adult goats were used for the experiment, no matter they were male or female, and the weight of the goats were 15-20 kg. The 10 mL marrow was obtained from the iliac crest of each goat. The BMSCs was expanded and passaged conventionally. The 3th BMSCs was infected by Adv-rhBMP-2 at 200 multiplicity of infection (MOI). The 1 x 10(8) infected BMSCs were digested by 0.25% trypsin and collected, then mixed with HA. The right tibia lengthening models were developed, and mixture with BMSCs was injected in the osteotomy position. The goats were divided randomly into 4 groups according to the material injected in operation, group A: Adv-rhBMP-2/BMSCs/HA (n = 6); group B: Adv-rhBMP-2/BMSCs (n = 5); group C: Adv-beta-gal/BMSCs/HA (n = 4); group D: sham without any injections (n = 4). After a seven-day latency period following ostectomy, distraction was carried out at a rate of 1 mm/day for 4 weeks. Roentgenography was practiced in 5, 8 and 12 weeks. After 12 weeks, the goats were sacrificed and dual-energy X-ray absorptiometry (DXA), biomechanical test and histology results were studied. RESULTS: After five and eight weeks surgery, X-ray test showed the distraction callus was more in group A and B than group C and D, and the radiographic score was significantly higher in group A and B than in the other two groups (P < 0.05); after 12 weeks surgery, the continued callus was formed in the distraction defects in all groups. DXA showed the mean bone mineral content of distraction callus in group A, B, C, D was (4.175 +/- 1.921), (2.600 +/- 0.638), (2.425 +/- 0.826) and (1.175 +/- 0.574) g, and the mean bone mineral density was (0.612 +/- 0.196), (0.630 +/- 0.159), (0.450 +/- 0.166) and (0.266 +/- 0.113) g/cm2. The group A and B was significantly higher than group C and D (P < 0.05). Biomechanical test showed the maximum loading of group A, B, C, D was (490.20 +/- 155.08), (350.59 +/- 80.48), (221.95 +/- 68.79) and (150.65 +/- 92.29) N, and elastic modulus was (178.24 +/- 105.80), (105.88 +/- 27.09), (81.18 +/- 48.67) and (50.35 +/- 47.64) MPa. The group A was significantly higher than in group C and D (P < 0.05). Histology observation revealed abundant bone formation in the distraction defects in group A, and the bone trabecula was arranged longitudinal and netlike. Histomorphology analysis revealed the bone volume in group A, B, C, D was 72.35% +/- 5.68%, 67.58% +/- 7.42%, 49.63% +/- 4.87% and 38.87% +/- 2.35%, and the bone formation was significantly greater in group A compared with group D (P < 0.05). CONCLUSION: HA mixed with rhBMP-2 modified BMSCs can accelerate distraction osteogenesis in goats.

Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2.

In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a beta-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.

[Effects of impaction on tissue engineered bone modified by BMP-2 gene].

OBJECTIVE: To observe effects of the direct impaction on the cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. METHODS: The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. RESULTS: There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappeared and there was much debris on the section. Some of the cells died and separated from the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the Adv-hBMP-2 modified tissue-engineered bone. CONCLUSION: The simulated impaction can decrease the cells survival and the bone formation of the Adv-hBMP-2 modified tissue-engineered bone. The survival cells still function well. It is feasible to use the tissue engineered bone in the impaction graft.

[Study on the differentiation of marrow mesenchymal stem cells of the rats mediated by bone morphogenetic protein 2].

OBJECTIVE: To explore the effect of age and gene therapy on the differentiation of marrow mesenchymal stem cells (MSCs) of the rats. METHODS: MSCs from the young (1-month-old), adult (9-month-old), and the aged (24-month-old) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase (ALP), collagen I (Col I), bone sialoprotein (BSP) and osteopontin (OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. RESULTS: There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col- I, OPN and BSP among the 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice among the 3 groups. CONCLUSION: MSCs obtained from the aged rats can restore their osteogenic activity following human BMP-2 gene transduction,therefore provides an alternative to treating the aged bone disease.

Immune response and effect of adenovirus-mediated human BMP-2 gene transfer on the repair of segmental tibial bone defects in goats.

BACKGROUND: Tissue-engineered bone may be used for filling bone defects. There are, however, no reports on this technique used in large animals. METHODS: We evaluated the effectiveness of, and immune response in repairing diaphyseal bone defects by gene transfer using bone morphogenetic proteins (BMPs). We used adenovirus-mediated human BMP-2 (Adv-hBMP-2) gene-transduced bone marrow stromal cells (BMSCs) to repair 2.1-cm segmental tibial bone defects in goats (group I, n = 7). An Adv-ssgal-transduced BMSC group (group II, n = 5), a non-transduced BMSC group (group III, n = 5), and an untreated group (group IV, n = 2) were used as controls. Self-secreted extracellular matrix was used as cellular carrier. RESULTS: Radiographic and histomorphometric examination demonstrated more callus in the bone defects of group I compared to other groups. Week 24 after implantation, the defect healing rates of groups I, II, III, and IV were 6/7, 1/5, 2/5, and 0/2, respectively. The maximum compressive strength of new tissue in the bone defects of group I was higher than those of groups II and III. Temporary cellular and persistent humoral immune responses against adenovirus were detected after hBMP-2 gene transfer. INTERPRETATION: We found that Adv-hBMP-2 genetransduced BMSCs had superior osteoinductivity in the repair of tibial bone defects in goats, but it could cause temporary cellular and persistent humoral immune responses against adenovirus.

Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue.

STUDY DESIGN: Experimental spinal cord injury using a rat model. OBJECTIVE: To investigate the potential for survival and migration of transplanted mesenchymal stem cells through the subarachnoid space into injured thoracic spinal cord tissue following injection into the more caudal lumbar spine. METHODS: A total of 70 adult Lewis rats were used with 64 having a partial and complete thoracic spinal cord injury (SCI) performed by the weight drop method at T9-T10 using the NYU Impactor. Six rats received only laminectomy for sham control. Mesenchymal stem cells (MSCs) were harvested from the femur of these rats and labeled by transduction of ANOVA virus containing green fluorescent protein (GFP) gene (Adv-GFP). At day 3, 5, and 7 after thoracic SCI, the rats received an injection into the subarachnoid space. The injections including: GFP-MSC, B-Gal-MSC, and PBS only. Injured rat spinal cords where harvested at day 7, 14, or 28, prepared for frozen sectioning, staining, and immunostaining. RESULTS: Adv-GFP transduced MSCs demonstrated strong green fluorescence both in the nucleus and in the cell body. Green fluorescent cells proven to be genuine GFP-positive cells were observed on the surface of the injured spinal cord parenchyma. The rate of the GFP-positive cells gathered into the central lesion within 10 mm was significantly higher than sham control. Also, GFP-positive cells were observed in the deeper area of the perivascular spaces, and some of them had integrated into the parenchyma. Immunostaining against Nestin demonstrated that some GFP-positive cells differentiated into neural stem cells and mature neurons or glial cells. CONCLUSIONS: Transplanted MSCs injected into the subarachnoid space of the lumbar spine can migrate to injured thoracic spinal cord tissue. The ratio of MSCs observed at the injury site was significantly higher than in the intact spinal cord, and also infiltrated into the deeper spinal cord parenchyma by the perivascular spaces. Lastly, some MSCs differentiated into Nestin-positive, immature neurons or glial cells.