Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration.

There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2- and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative.

Outcome of resection of a symptomatic os trigonum.

BACKGROUND: While an os trigonum at the posterolateral aspect of the talus is usually asymptomatic, this inconsistently present accessory bone has been associated with persistent posterior ankle pain, which has been described as the os trigonum syndrome. We present the clinical results of excision of the os trigonum through a posterolateral approach and report several factors affecting the clinical outcome. METHODS: During a five-year period from 1994 through 1999, forty-one patients had a failure of nonoperative treatment of os trigonum syndrome and underwent excision of a symptomatic os trigonum. In all cases, the os trigonum syndrome was diagnosed on the basis of the history, physical examination, and radiographs. Postoperatively, the patients were evaluated according to the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale. A questionnaire was used to evaluate the effect of several factors on the clinical outcome. RESULTS: The average duration of follow-up was forty-four months. The postoperative AOFAS score averaged 87.6 points. The thirty-three patients who had had symptoms for two years or less prior to the surgery had an average postoperative score of 90 points compared with 78 points for the eight patients who had had preoperative symptoms for more than two years (p = 0.011). Eight patients had sural nerve sensory loss, which was temporary in four and permanent in four. A superficial wound infection developed in one patient, and reflex sympathetic dystrophy developed in another. CONCLUSIONS: An os trigonum that is persistently symptomatic after a minimum three-month trial of nonoperative treatment can be excised through a posterolateral approach with highly satisfactory results. The main complication of this procedure is sural nerve injury.