R. Bruce Rutherford

Gene Therapy-Directed Osteogenesis: BMP-7-Transduced Human Fibroblasts Form Bone in Vivo

An ex vivo gene therapy strategy was used to achieve localized skeletal regeneration in vivo. When an adenovirus vector engineered to express bone morphogenetic protein 7 transduced human gingival fibroblasts or rat dermal fibroblasts, these nonosteogenic tissues formed bone and supported the development of hematopoietic tissue when transplanted into immunocompromised mice. Transduced gingival fibroblasts formed marrow-containing ossicles in 100% of transplants after 1-2 weeks in vivo (n = 30). Immunostaining with murine and human-specific antisera raised against osteonectin and in situ hybridization of human-specific Alu genomic sequence demonstrated that the newly formed bone organ was a chimera of both the human donor and the mouse recipient cells. In experiments of greater clinical relevance, AdCMVBMP-7-transduced dermal fibroblasts repaired critical size skeletal defects in rat calvariae (n = 6). The results of this study suggest a bifunctional role of BMP-7-transduced fibroblasts. The transduced, nonosteogenic cells not only secreted biologically active BMP-7 in vitro and in vivo, but also differentiated into bone-forming cells in vivo. This model exploits the use of an easily biopsied, self-regenerating tissue such as gingiva or skin and suggests that local regeneration of tissues by ex vivo gene therapy may not require that autogenous cells be cultured from the tissue that is to be regenerated.

Gene therapy for bone formation: In vitro and in vivo osteogenic activity of an adenovirus expressing BMP7

Bone morphogenetic proteins (BMPs) are well‐established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half‐life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone‐inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV‐BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842–1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV‐BMP7 produced high levels of BMP‐7 (approximately 0.5 μg/106 cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP‐7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV‐BMP7 was mixed with a bovine bone‐derived collagen carrier (108 plaque‐forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV‐BMP7 transduced cells produce biologically active BMP‐7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration. J. Cell. Biochem. 78:476–486, 2000.

Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo.

Experimental cell or ex vivo gene therapy for localized bone formation typically uses osteoprogenitor cells propagated from periosteum or bone marrow. Both require bone or marrow biopsies to obtain cells. We have demonstrated that implantation of gingival or dermal fibroblasts transduced with BMP ex vivo, using a recombinant adenovirus (AdCMVBMP) attached to porous biodegradable scaffolds, form bone in vivo. Here we show that BMP-7-transduced fibroblasts suspended in injectable thermoset hydrogels form complete ossicles on subcutaneous injection and repair segmental defects in rat femurs. Bone formation was preceded by an intermediate cartilage stage. To determine the fate of the implanted transduced cells, thermoset hydrogel suspensions of ex vivo BMP-7-transduced or nontransduced fibroblasts were placed in diffusion chambers and implanted to allow development in vivo without direct contact with host cells. Only the BMP-transduced fibroblasts formed bone within the diffusion chambers in vivo, revealing that BMP transduction induces osteoblastic conversion of these cells.

Gene Therapy Approaches for Bone Regeneration

Gene therapy represents a promising approach for delivering regenerative molecules to specific tissues including bone. Several laboratories have shown that virus-based BMP expression vectors can stimulate osteoblast differentiation and bone formation in vivo. Both in vivo and ex vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Adenovirus and direct DNA delivery of genes encoding regenerative molecules can heal critical-sized defects of cranial and long bones. Although osteogenic activity can be demonstrated for individual BMP vectors, substantial synergies may be achieved using combinatorial gene therapy to express complimentary osteogenic signals including specific combinations of BMPs or BMPs and transcription factors. Further control of the bone regeneration process may also be achieved through the use of inducible promoters that can be used to control the timing and magnitude of expression for a particular gene. Using these types of approaches, it should be possible to mimic natural processes of bone development and fracture repair and, in so doing, be able to precisely control both the amount and type of bone regenerated.

Expression of genes for bone morphogenetic proteins and receptors in human dental pulp

Bone morphogenetic proteins (BMP) have been shown to induce reparative dentine formation experimentally but the cells responsible, which respond to BMPs, have not been identified. The BMP signal is probably mediated by interaction of type I and II BMP receptors (R). Here, the RNA of human adult dental pulp and pulp cells in culture was examined by reverse transcription (RT) polymerase chain reaction (PCR) for evidence of mRNA for BMPs. mRNAs for BMP-2, -4, osteogenic protein-1, ActR-1 (activin-like kinase receptor), BMPR-IA, -IB and -II were detected by RT-PCR. The 698-bp PCR fragment for BMPR-IB was used to probe pulp cells for expression of that receptor. Cell expression of BMPR-IB was detected by the hybridization probe. The findings suggest that resident pulp cells may be able to respond to BMPs to initiate tissue formation.

Ex Vivo Gene Therapy for Skeletal Regeneration in Cranial Defects Compromised by Postoperative Radiotherapy

Because radiation remains a common postoperative treatment for head and neck cancers, it is critical to determine whether new bone-regenerative approaches are effective for healing craniofacial defects challenged by therapeutic doses of radiation. The objective of this study was to determine whether the deleterious effects of radiotherapy could be overcome by ex vivo gene therapy to heal craniofacial defects. Rat calvarial critical-sized defects were treated with either an inlay calvarial bone graft or syngeneic dermal fibroblasts transduced ex vivo with an adenovirus engineered to express bone morphogenetic protein 7 (BMP-7), a morphogen known to stimulate bone formation. Two weeks postoperatively, either no radiation or a single 12-Gy radiation dose was delivered to the operated area and the tissue was harvested 4 weeks later. None of the inlay bone grafts healed at the wound margins of either the radiated or nonradiated sites. In contrast, bone was successfully regenerated when using an ex vivo gene therapy approach. More bone formed in the nonradiated group as determined by the percentage of defect surface covered (87 +/- 4.1 versus 65 +/- 4.7%; p = 0.003) and percentage of defect area filled by new bone (60 +/- 5.9 versus 32 +/- 2.7%; p = 0.002). Although the effects of radiation on the wound were not completely overcome by the gene therapy approach, bone regeneration was still successful despite the radiation sensitivity of the fibroblasts. These results indicate that BMP-7 ex vivo gene therapy is capable of successfully regenerating bone in rat calvarial defects even after a therapeutic dose of radiation. This approach may represent a new strategy for regenerating skeletal elements lost due to head and neck cancer.

Bone Regeneration in Cranial Defects Previously Treated with Radiation

Objectives/Hypothesis: Bone reconstruction in the head and neck region is frequently performed in the context of previous radiation treatment. Thus, the effectiveness of tissue engineering approaches for regenerating bone in radiated defects needs to be determined before considering application to patients. Incomplete healing is described when using osteoinductive protein therapy alone for bone defects previously treated with radiation. We hypothesized that a different approach using ex vivo gene therapy can heal these severely compromised defects.

Early Events: The In Vitro Conversion of BMP Transduced Fibroblasts to Chondroblasts

Strategies for localized skeletal regeneration using either cell or ex vivo gene transfer typically utilize preosteoblasts from bone or bone marrow biopsies. Our studies have demonstrated than ex vivo bone morphogenetic proteins (BMP) transduced fibroblasts convert to osteoblasts and form bone in vivo [1, 2]. In addition when suspended in a variety of thermoset hydrogels, these cells are capable of ectopic or orthotopic bone formation. To study the mechanism of the phenotypic conversion of BMP transduced fibroblasts, we have initiated characterization of three-dimensional (3D) cultures derived from these cell/collagen hydrogel composites. These data reveal that the BMP, but not control transduced fibroblasts, secrete BMP-7 and acquire some chondroblastic traits in 3D cultures. These traits include altered cell shape and changes in the extracellular matrix such as accumulation of cartilage proteoglycan, type II collagen, and mineral deposition by 3 weeks. These studies suggest that this culture system may be useful for elucidating early mechanistic events in the BMP-induced conversion of fibroblasts to osteoblasts.