Volker M. Betz

Direct Percutaneous Gene Delivery to Enhance Healing of Segmental Bone Defects

BACKGROUND Healing of segmental bone defects can be induced experimentally with genetically modified osteoprogenitor cells, an ex vivo strategy that requires two operative interventions and substantial cost. Direct transfer of osteogenic genes offers an alternative, clinically expeditious, cost-effective approach. We evaluated its potential in a well-established, critical-size, rat femoral defect model. METHODS A critical-size defect was created in the right femur of forty-eight skeletally mature Sprague-Dawley rats. After twenty-four hours, each defect received a single, intralesional, percutaneous injection of adenovirus carrying bone morphogenetic protein-2 (Ad.BMP-2) or luciferase cDNA (Ad.luc) or it remained untreated. Healing was monitored with weekly radiographs. At eight weeks, the rats were killed and the femora were evaluated with dual-energy x-ray absorptiometry, micro-computed tomography, histological analysis, histomorphometry, and torsional mechanical testing. RESULTS Radiographically, 75% of the Ad.BMP-2-treated femora showed osseous union. Bone mineral content was similar between the Ad.BMP-2-treated femora (0.045 +/- 0.020 g) and the contralateral, intact femora (0.047 +/- 0.003 g). Histologically, 50% of the Ad.BMP-2-treated defects were bridged by lamellar, trabecular bone; the other 50% contained islands of cartilage. The control (Ad.luc-treated) defects were filled with fibrous tissue. Histomorphometry demonstrated a large difference in osteogenesis between the Ad.BMP-2 group (mean bone area, 3.25 +/- 0.67 mm(2)) and the controls (mean bone area, 0.65 +/- 0.67 mm(2)). By eight weeks, the Ad.BMP-2-treated femora had approximately one-fourth of the strength (mean, 0.07 +/- 0.04 Nm) and stiffness (mean, 0.5 +/- 0.4 Nm/rad) of the contralateral femora (0.3 +/- 0.08 Nm and 2.0 +/- 0.5 Nm/rad, respectively). CONCLUSIONS A single, percutaneous, intralesional injection of Ad.BMP-2 induces healing of critical-size femoral bone defects in rats within eight weeks. At this time, the repair tissue is predominantly trabecular bone, has normal bone mineral content, and has gained mechanical strength.

Delayed administration of adenoviral BMP-2 vector improves the formation of bone in osseous defects

The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague–Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 × 108 plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.

Bone tissue engineering and repair by gene therapy.

Many clinical conditions require the stimulation of bone growth. The use of recombinant bone morphogenetic proteins does not provide a satisfying solution to these conditions due to delivery problems and high cost. Gene therapy has emerged as a very promising approach for bone repair that overcomes limitations of protein-based therapy. Several preclinical studies have shown that gene transfer technology has the ability to deliver osteogenic molecules to precise anatomical locations at therapeutic levels for sustained periods of time. Both in-vivo and ex-vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Genetic engineering of adult stem cells from various sources with osteogenic genes has led to enhanced fracture repair, spinal fusion and rapid healing of bone defects in animal models. This review describes current viral and non-viral gene therapy strategies for bone tissue engineering and repair including recent work from the authors laboratory. In addition, the article discusses the potential of gene-enhanced tissue engineering to enter widespread clinical use.

Healing of Large Segmental Bone Defects Induced by Expedited Bone Morphogenetic Protein-2 Gene-Activated, Syngeneic Muscle Grafts

Numerous preclinical studies have shown that osseous defects can be repaired by implanting bone morphogenetic protein (BMP)-2-transduced muscle cells. However, the drawback of this treatment modality is that it requires the isolation and long-term (approximately 3 weeks) culture of transduced autologous cells, which makes this approach cumbersome, time-consuming, and expensive. Therefore, we transferred BMP-2 cDNA directly to muscle tissue fragments that were held in culture for only 24 hr before implantation. We evaluated the ability of such gene-activated muscle grafts to induce bone repair. Two of 35 male, syngeneic Fischer 344 rats used in this study served as donors for muscle tissue. The muscle fragments remained unmodified or were incubated with an adenoviral vector carrying the cDNA encoding either green fluorescent protein (GFP) or BMP-2. Critical-size defects were created in the right femora of 33 rats and remained untreated or were filled (press fitted) with either unmodified muscle tissue or GFP-transduced muscle tissue or with BMP-2-activated muscle tissue. After 6 weeks, femora were evaluated by radiography, microcomputed tomography (muCT), histology, and biomechanical testing. Six weeks after implantation of BMP-2-activated muscle grafts, 100% of the bone defects were bridged, as documented by radiographs and muCT imaging, and showed formation of a neocortex, as evaluated by histology. Bone volumes of the femora repaired by BMP-2-transduced muscle were significantly (p = 0.006) higher compared with those of intact femora and the biomechanical stability was statistically indistinguishable. In contrast, control defects receiving no treatment, unmodified muscle, or GFP-transduced muscle did not heal. BMP-2 gene-activated muscle grafts are osteoregenerative composites that provide an expedited means of treating and subsequently healing large segmental bone defects.

BMP-2 gene activated muscle tissue fragments for osteochondral defect regeneration in the rabbit knee.

Previously published data indicate that BMP‐2 gene activated muscle tissue grafts can repair large bone defects in rats. This innovative abbreviated ex vivo gene therapy is appealing because it does not require elaborative and time‐consuming extraction and expansion of cells. Hence, in the present study, we evaluated the potential of this expedited tissue engineering approach for regenerating osteochondral defects in rabbits.

The effect of BMP-7 gene activated muscle tissue implants on the repair of large segmental bone defects

BACKGROUND This study was conducted in order to investigate the effect of Bone Morphogenetic Protein-7 (BMP-7) transduced muscle cells on bone formation and to further develop an innovative abbreviated ex vivo gene therapy for bone repair. As conventional ex vivo gene therapy methods require an elaborative and time-consuming extraction and expansion of cells we evaluated an expedited approach. Fragments of muscle tissue were directly activated by BMP-7 cDNA and implanted into bone defects. METHODS 25 male, syngeneic Fischer 344 rats were used in the present study. Muscle tissue was harvested from two donor rats and either transduced with an adenovirus carrying the BMP-7 cDNA or remained unmodified. 5mm osseous defects in the right femora of 23 rats were treated with either unmodified muscle tissue (control group) or BMP-7 activated muscle tissue (treatment group). Six weeks after surgery, rat femora were evaluated by radiographs, micro-computed tomography (μCT) and histology. RESULTS Implantation of BMP-7 activated muscle grafts led to bony bridging in 5 out of 12 defects (41.7%) and to bone formation without bridging in 2 out of 12 defects. In 2 femoral defects of this group radiographs, μCT-imaging and histology did not reveal significant mineralization. Three animals of the BMP-7 treatment group had to be euthanized due to serious wound infection. The bone volume of the treatment group was significantly (p=0.007) higher compared to the control group. CONCLUSION This study shows that BMP-7 gene activated muscle fragments have the potential to regenerate critical-size segmental bone defects in rats. However, further development of this promising expedited treatment modality is required to improve the healing rate and to investigate if the high infection rate is related to treatment with BMP-7 activated muscle grafts.

Gene activated tissue grafts for sustained bone morphogenetic protein-2 delivery and bone engineering: is muscle with fascia superior to muscle and fat?

Previously, we have presented an expedited strategy for sustained delivery of bone morphogenetic protein‐2 (BMP‐2) to bone lesions based on the implantation of gene‐activated fat and muscle fragments. The aim of the present in vitro experiments was to evaluate the potential of muscle with fascia as a BMP‐2 delivering osteo‐regenerative implant in comparison to fat tissue and muscle alone. Subcutaneous fat, muscle, and muscle with fascia were harvested from Fischer 344 rats. The tissues were cut into small pieces and cultured for up to 90 days after direct transduction with adenoviral BMP‐2 or green fluorescence protein vectors. Different vector doses were applied, and proliferation, long‐term BMP‐2 production, and osteogenic differentiation of the 3 different tissues were investigated in vitro. Muscle with fascia produced the largest amounts of BMP‐2. Expression of the transgene was detected for up to 90 days. Proliferation was reduced with increased vector doses. Muscle with fascia showed a higher potential for osteogenic differentiation than fat, but it was not improved as compared to muscle alone. A dose of 4 × 108 plaque forming units of the adenoviral BMP‐2 vector appeared to be the optimal dose for transduction of muscle with fascia. Because muscle with fascia produced higher amounts of BMP‐2 as compared to muscle alone or fat tissue grafts, showing a high potential for osteogenic differentiation, it might represent an improved osteo‐regenerative implant facilitating endogenous repair. Future studies should investigate the effect of muscle with fascia transduced with 4 × 108 plaque forming units on bone healing in vivo.

An expedited approach for sustained delivery of BMP‐7 to bone defects using gene activated fragments of subcutaneous fat

Delivery of bone morphogenetic protein‐7 (BMP‐7) to bone defects can be improved by applying gene transfer methods. However, traditional ex vivo gene therapy approaches are cumbersome and costly, requiring the extraction and culturing of cells. Therefore, we evaluated a novel, expedited ex vivo BMP‐7 gene transfer technology based on the use of fragments of subcutaneous fat tissue.

An expedited approach for sustained delivery of bone morphogenetic protein-7 to bone defects using gene activated fragments of subcutaneous fat

Delivery of bone morphogenetic protein‐7 (BMP‐7) to bone defects can be improved by applying gene transfer methods. However, traditional ex vivo gene therapy approaches are cumbersome and costly, requiring the extraction and culturing of cells. Therefore, we evaluated a novel, expedited ex vivo BMP‐7 gene transfer technology based on the use of fragments of subcutaneous fat tissue.

Osteoinduction within BMP-2 transduced muscle tissue fragments with and without a fascia layer: implications for bone tissue engineering

Bone can be engineered in vivo by implantation of gene-activated muscle tissue fragments. This expedited approach may be further improved by use of muscle tissue with attached fascia. The aim of this in vitro study was to provide an in depth comparison of the osteogenic differentiation capacity of muscle alone and muscle with fascia after BMP-2 transduction. Skeletal muscle tissue from rats was cut into pieces with and without a fascia layer on the surface. Adenoviral BMP-2 or GFP vectors were used for transduction. Osteogenic differentiation within the tissue fragments was evaluated and compared by qRT-PCR, alizarin red S staining, histomorphometry and immunohistology. Transduction efficiency and level of transgene expression were higher for muscle with fascia than muscle alone. Transduction with BMP-2 led to a significant upregulation of bone marker genes, proteins, and calcium deposition in both groups. Interestingly, histological evaluation revealed that osteoinduction did not occur within the fascia layer itself. The upregulation of bone marker genes in muscle with fascia was significantly lower after 2 weeks but similar after 4 weeks of in vitro culture in comparison to muscle alone. The fascia layer led to higher transduction efficiency and enhanced BMP-2 expression. Despite fascia’s lower capacity for osteogenic differentiation, muscle implants may benefit from the fascia layer by the improved ability to deliver BMP-2. The presented data may contribute to the development of a novel, cost-effective, single-surgery bone engineering technology and encourage the evaluation of the osteoregenerative potential of muscle with fascia in an animal model.