Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats.

The efficacy of beta-tricalcium phosphate (beta-TCP) loaded with bone morphogenetic protein-2 (BMP-2)-gene-modified bone-marrow mesenchymal stem cells (BMSCs) was evaluated for the repair of experimentally-induced osteonecrosis of the femoral head in goats. Bilateral early-stage osteonecrosis was induced in adult goats three weeks after ligation of the lateral and medial circumflex arteries and delivery of liquid nitrogen into the femoral head. After core decompression, porous beta-TCP loaded with BMP-2 gene- or beta-galactosidase (gal)-gene-transduced BMSCs was implanted into the left and right femoral heads, respectively. At 16 weeks after implantation, there was collapse of the femoral head in the untreated group but not in the BMP-2 or beta-gal groups. The femoral heads in the BMP-2 group had a normal density and surface, while those in the beta-gal group presented with a low density and an irregular surface. Histologically, new bone and fibrous tissue were formed in the macropores of the beta-TCP. Sixteen weeks after implantation, lamellar bone had formed in the BMP-2 group, but there were some empty cavities and residual fibrous tissue in the beta-gal group. The new bone volume in the BMP-2 group was significantly higher than that in the beta-gal group. The maximum compressive strength and Young's modulus of the repaired tissue in the BMP-2 group were similar to those of normal bone and significantly higher than those in the beta-gal group. Our findings indicate that porous beta-TCP loaded with BMP-2-gene-transduced BMSCs are capable of repairing early-stage, experimentally-induced osteonecrosis of the femoral head and of restoring its mechanical function.

BMP2 gene therapy on the repair of bone defects of aged rats.

Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Ialpha1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/beta-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.

Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone.

Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteo-conductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.

Ptosis surgery on chronic myasthenia gravis.

We report three cases of long-standing, early onset myasthenia gravis. The eyes of all three patients eventually became disabled, and the patients were almost unable to move their eyes in any direction. We termed this condition as "frozen eyes" appearance. We discuss the unique clinical condition and follow the electromyographic and neuro-ophthalmological characteristics. The patients all suffered from ptosis, mild to moderate exotropia, and facial and oropharyngeal weakness. All of the patients responded to the neostigmine test positively at the early stage. Two of them were seropositive. The results of electromyography for two of these three cases were abnormal. The average duration of follow-up was 5 years. The treatments included conventional recession and resection procedures with adjustable sutures for strabismus and frontalis suspension for blepharoptosis. The patients achieved stable satisfactory results in over one-and-a-half years of postoperative follow up.