*Enhancement of Drug Delivery by Hypertonic Saline Solution Following Spinal Cord Injury
W.F. Young, MD; R.F. Tuma, PhD; U.S. Vasthare, PhD
Gene Expression During Posterolateral Lumbar Spine Fusion: Effect of BMP-2
S.D. Boden, MD; M.A. Morone, MD, PhD; G. Martin, MD; G. Hair, PhD; M. Racine
Introduction. Posterolateral spine arthrodesis may result in failure to achieve a solid bony union in up to 35% of patients. Recent studies using a validated rabbit model have characterized the healing process using quantitative histomorphometry. However, little is known from a molecular biology perspective about expression of bone and cartilage-related genes during this process. In addition, the role of bone morphogenetic proteins is unclear. Using current molecular biology techniques reverse transcription/polymerase chain reaction (RT/PCR), small quantities of tissue can be used to study the expression of specific genes. The goals of this investigation were (1) to describe the temporal and spatial pattern of bone and cartilage-related gene expression; (2) to correlate the gene expression patterns with the histologic healing patterns; and (3) to describe the effect of bone morphogenetic protein-2 (BMP-2) on gene expression during spine fusion healing.
Materials and Methods. Part I. After approval by the IACUC, twenty adult New Zealand white rabbits underwent L4-5 posterolateral intertransverse process spine arthrodesis using autogeneous iliac crest bone graft. Rabbits were euthanized at 2 days, 4 days, 1, 2, 3, 4, 5, 6, or 10 weeks following surgery.
Part II. Fourteen adult New Zealand white rabbits underwent arthrodesis using autogeneous iliac crest bone that had been soaked in 1/5 mg/ml rhBMP-2 solution (Genetics Institute, Cambridge, MA). Rabbits were euthanized at the same time points as above. RNA Extraction. Fusion masses were harvested and divided into thirds (two transverse process outerzones and one central zone). Each third was then frozen in liquid nitrogen. Total RNA was extracted using 4 M guanidine isothiocyanate. RNA was extracted from iliac crests harvested from four animals to serve as a control for baseline gene expression at time 0 in the fusion mass. The RNA then underwent RT/PCR to study the expression of various genes. Unique PCR primers were designed using sequence information available in Genebank. The number of PCR cycles was 22 for bone morphogenetic protein-2 (BMP-2), BMP-4, BMP-6, Type I collagen, Type II collagen, osteopontin, osteonection, osteocalcin, alkaline phosphatase and GAPDH (a housekeeping gene). All PCR products were separated on a 12% acrylamide gel and exposed on a phosphorimager. The intensities were normalized to that of GAPDH and then normalized to the relative intensity of each mRNA PCR product at time zero. Quantitation was performed on 2-3 PCR replicates of three independent samples from separate RT reactions and expressed as mean + standard error of the mean (SEM). A one-way analysis of variance (ANOVA), with Bonferroni’s post hoc multiple comparison test, was used to detect differences at different time points. All findings described below reached statistical significance defined as p<.05.
Results. Part I. A unique temporal and spatial pattern of osteoblast-related gene expression was observed based on RT/PCR analysis of RNA from the different zones of the fusion mass. During the second and third weeks, a significant increase was seen in Type I collagen gene expression. Osteopontin (OP) and osteonectin (ON) were both increased by one week, with osteopontin peaking in week three (150-fold increase) and osteonectin in week two (175-fold increase). A 28-fold increase in osteocalcin PCR product expression was seen in weeks three to four in the outer zones. Gene expression in the central zone of the fusion mass lagged one to two weeks behind that of the two outer zones correlating with the central lag effect previously observed in the histologic healing sequence. Expression of several BMPs was also studied. In the outer zones, increased BMP-2 expression was seen in weeks two to six with peak expression in weeks three to four (40-fold increase). BMP-4 demonstrated a different pattern with a 40-fold increase in week one that decreased significantly by week three. BMP-6 had an early increase on day two (54-fold) and a second peak (100-fold) in weeks four to five. These findings suggest unique time patterns of expression and possibly unique roles for various BMPs during spine fusion.
Part II. Soaking the autogenous bone graft with rhBMP-2 had a dramatic impact on the gene expression during spine fusion healing. Most notably, there was a significantly greater increase in BMP-6 expression on day two (95-fold) in the outer zone and an earlier second BMP-6 peak in the central zone at week two (instead of week four). In addition, expression of other bone-related genes described in Part I was seen earlier and at higher levels in the presence of rhBMP-2. The previously observed lag effect in the central zone was minimized with the addition of rhBMP-2 that may explain the decreased nonunion rate described in previous animal studies of spine fusion using rhBMP-2.
Discussion. We have shown that it is possible to reproducibly measure gene expression in spine fusion masses using RT/PCR technology. BMP-6 was the earliest of the BMPs to show increased expression and may be a critical factor in the initiation of spine fusion healing. Soaking of autograft with rhBMP-2 increased the early BMP-6 peak and decreased the central lag effect that may explain why fusions with rhBMP-2 heal faster with less nonunion. In addition to helping understand how rhBMP-2 may function in vivo, these baseline gene expression data will facilitate the design of experiments with fusions enhanced (ultrasound, electrical stimulation) or retarded (nicotine, NSAIDs) to elucidate the potential mechanisms of action of these agents and to design gene-specific biologic strategies to more effectively manipulate the spine fusion healing process.
Localization, Reproducibility and Dosimetry for Spinal Radiosurgery
A.A.F. DeSalles, MD, PhD; P.M. Medin, MS; T.D. Solberg, PhD; C.H. Cagnon; M.T. Selch; J.P. Johnson; J.B. Smathers; E.R. Cosmon
Purpose. A new method for fractionated stereotactic irradiation of spinal malignancies has been investigated in preparation for clinical trials. Evaluations of the theoretical and practical limitations of localization accuracy and the implementation of the method in swine are presented.
Methods and Materials. In a percutaneous procedure, a minimum of three small (1.7mm diameter) titanium markers are permanently affixed to the bone of the vertebral column. Markers are localized on biplanar radiographs. Isocenter positions are determined on computed tomography (CT) relative to the rigid body defined by the vertebral markers. The markers provide the means for accurate day-to-day reproducibility necessary for fractionated radiation delivery. A special frame defines a coordinate system inside which the patient rests. A three-dimensional reference frame through the patient is established using external fiducial markers in a localization bridge. Radiographs coupled with a rigid body rotation algorithm account for daily differences in patient position relative to the stereotactic frame; therefore, it is not important if a patient moves between treatments. Immobilization is utilized to ensure movement is restricted between the time of the daily radiographs and the completion of radiation delivery. A treatment simulation program was written to estimate the uncertainty inherent in the localization technique. Phantom studies were used to verify theoretical uncertainty calculations. A swine model was used to evaluate the difficulty and duration of the implant technique, the suitability of the vertebral processes as an implant site, vertebral motion due to normal respiration and the ability to target one vertebra with markers in an adjacent vertebra.
Results. Theoretical accuracy studies confirmed that localization accuracy is a function of marker separation. Phantom studies involving 296 measurements showed that individual implants could be localized within ±0.25 mm. The largest targeting error observed in 3,600 measurements of one hundred clinically relevant implant configurations was 1.17 mm. The implant procedure took 5-10 minutes per site. No significant migration of implants was observed out to thirty-five days post-implantation and respiratory motion had no detectable influence on vertebral position. Adjacent vertebrae may be useful for targeting one another with a small sacrifice in localization accuracy.
Conclusions. The use of implanted markers for localization of spinal malignancies has merit for applications in stereotactic radiotherapy. Phantom measurements suggest localization accuracy similar to intracranial stereotactic radiotherapy techniques is achievable. Swine studies suggest that the implant technique is expedient and feasible for tumor targeting purposes.
*Spine Fusion With Tissue Engineered Scaffolds
K.J. Kopacz, MD; K.S. James, PhD; J.R. Parsons, Jr., PhD; J.B. Kohn, PhD
The specific aim of this project is to develop and test a tissue-engineering concept for spine fusion. It was hypothesized that a degradable polymeric matrix could act as a scaffold for generating a mineralized and vascularized bone graft to enhance the fusion process. The polymeric matrix is fabricated from poly (DTE carbonate), a degradable polymer system based on the amino acid tyrosine. This material is a promising alternative to the commonly investigated poly (alpha hydroxyl ester) such as poly (lactic acid) and poly (glycolic acid), for they exhibit excellent long-term biocompatibility and a superior interface with bone.
We initiated the project by performing a pilot study consisting of four animals carried out to two months to (a) assure we could duplicate the intertransverse spine fusion model of Boden et al., and (b) establish baseline biocompatibility data of poly (DTE carbonate) scaffolds at the site of bone grafting.
The rabbits tolerated the surgical procedure well. Autograft bone was harvested from the iliac crests. Each animal was fused at L5-6 with autograft bone on one side and poly (DTE carbonate) scaffolds on the other. Poly (DTE carbonate) scaffolds were also implanted subcutaneously. Each autograft site was fused by two months. This was confirmed by X-ray, manual palpation and histological analysis. In contrast, bony fusion was not observed with poly (DTE carbonate) scaffolds. Histological analysis revealed fibrovascular soft tissue ingrowths throughout the scaffolds. Bone ingrowths were seen into the scaffolds where they overlapped the decorticated transverse process. However, bone was not conducted along the length of the 2cm implant. It is important to note that no significant inflammatory response was evident inside or circa the implanted scaffold.
Having performed this pilot study, we came to appreciate the aggressiveness of the spine fusion mode, ie, the graft material must grown one across a 2/0-2/5cm gap between adjacent transverse processes. Given this and the finding that the poly (DTE carbonate) scaffold tested was not itself osteoconductive, we have decided to change the proposed experimental method. Rather than moving immediately to the spine fusion model, the well-characterized rabbit calvaria trephine defect model will be employed to screen for the most promising scaffold configuration to be ultimately evaluated in the spine fusion model. Our laboratory has prior experience with this model where it has been an effective means of comparing novel bone graft substitutes to autograft controls. This is a less technically demanding model than the spine fusion procedure. Consequently, experimental variability should be decreased.
The trephine defect screening study was recently initiated. The trephine defect model consists of two 8mm diameter defects in a rabbit’s skull. Two time points are being investigated: three weeks (bone bridging has been documented with autograft and demineralized bone matrix at two weeks) and eight weeks (the acute inflammatory response has subsided and full bone ingrowths and remodeling should be evident in an osteoconductive/inductive material). Florescent bone labels are used at two and four weeks to document the extent of bone ingrowths at intermediate time points. The specimens will be embedded in polymethylmethacrylate (PMMA), analyzed histologically and quantified via established histomorphometry techniques (extent of bone ingrowths and rate of bone ingrowths). Poly (DTE carbonate) scaffolds and those scaffolds augmented with growth factors and cells as described in the original proposal will be evaluated in this model. Experimental bone graft results are compared to autograft (positive) and empty (negative) control defects. Each experimental condition will be evaluated in five implant sites per time point.
We anticipate completing the trephine screening process with subsequent evaluation of the most promising tissue engineered bone graft in the spine fusion model commencing soon thereafter.
*Abstracts/permission forms not received at the time of publication
**Current and ongoing research