THORACOSCOPIC INTRADISCAL SPINE FUSION USING GENE THERAPY

KD Riew,
J Lou,
NM Wright,
S.–L Cheng,
T Bae,
LV Avioli
* (a – Washington University, Institute for Minimally Invasive Surgery)
Barnes–Jewish Hospital at Washington University School of Medicine St. Louis, MO, USA

INTRODUCTION:
Attempts to produce intradiscal fusion using BMP injections have not been successful. The purpose of the present study is to determine if gene therapy can be utilized to achieve anterior intradiscal fusion in pigs, without the use of bone or hardware.

METHODS:
Rib marrow derived mesenchymal stem cells (MSC) were isolated from each of 3 pigs and expanded in culture in vitro. The MSC were transduced with either adenoviruses carrying the gene for BMP–2 (Adv–BMP2) or bgalactosidase (Adv–bgal), as a control. In Vitro Study: BMP–2 protein expression in conditioned media was tested by immunoprecipitation and Western blot with a specific monoclonal antibody h4b2/5.10.24. Alkaline phosphatase activity and matrix mineralization (Von Kossa's stain) were determined. Protein expression of Type I collagen, osteopontin, and bone sialoprotein were analyzed by Western blot. In Vivo Study: Following general anesthesia, 4 thoracic disc spaces in each of 3 pigs were minimally prepared for cell implantation under thoracoscopic visualization. At each level, approximately 1 cc of disc material was removed to make room for the cells. The rest of the disc space was left undisturbed. In each pig, 2 discs were injected with autologous MSC transduced with Adv–BMP2. Two other discs were respectively implanted with Adv–bgal transduced MSC (control 1) and non–transduced MSC (control 2). The animals were sacrificed 6 weeks post implantation. Sagittal 2–D CT images were blindly assessed for fusion by a radiologist. Blinded histologic examination was performed on all specimens.

RESULTS:
In Vitro Study: A BMP–2 protein band was detected in the conditioned medium of Adv–BMP2 transduced MSC, but not the controls. Compared to the controls, Alkaline phosphatase activity of Adv–BMP2 transduced MSC increased 5 fold and matrix mineralization was induced. Consistent with the expression of functional BMP–2 protein, Adv–BMP2 transduced MSC increased protein expression of Type I collagen, osteopontin, and bone sialoprotein. In Vivo Study: Anterior spine fusion was demonstrated by radiographic examination in all 6 discs implanted with Adv–BMP2 transduced MSC. The 6 control discs implanted with either Adv–bgal or untreated MSC had little or no intervening bone. Histologic examination demonstrated bridging bone from endplate to endplate for all 6 discs implanted with Adv–BMP2 transduced MSC. The 6 control discs implanted with either Adv–bgal or untreated MSC had no bridging bone.

DISCUSSION/CONCLUSION:
To our knowledge, this is the first report of successful anterior spine fusion without the use of bone or hardware. With minimal disc preparation, the transduced MSC resulted in intradiscal fusion in 100% of the discs.

* If noted, the author indicates something of value received. The codes are identified as: a–research or institutional support, b–miscellaneous funding, c–royalties, d–stock options, e–consultant or employee.