Tissue Engineered Therapy To Heal Critical-Sized Rat Cranial Defects

Wayne Ozaki, MD, S.R. Winn, PhD, G. Xi, MD, C.C. Hung, MD, A. Sasaki, MD, Y. Hu, PhD

Background

The incidence of cleft lip and palate is approximately 1:1,000. Complete cleft lips and palates involve the alveolar processes of the maxilla. Alveolar clefts are usually grafted prior to eruption of the adult canine using cranial or iliac crest bone grafts. Grafting procedures yield a predictable success rate of approximately 80%. Despite a laudatory record, autologous bone grafting has recognized liabilities. These disadvantages include: a finite supply of donor tissue, increased donor site morbidity, protracted hospitalization, and increased costs. A tissue engineered BMP formulation could be a suitable alternative to bone grafting and reduce many of these clinical limitations.

Bone morphogenetic proteins (BMPs) are essential components in bone regeneration (Urist, 1989) and promote differentiation of pluripotential cells into bone-forming cells (Kingsley, 1994). Despite their proven preclinical efficacy, there are several distinct disadvantages of BMPs. They include: the need for very high dosing to achieve clinical success, potential systemic toxicities, short duration of action, and relatively high expense. To minimize these disadvantages, we used plasmid DNA encoding for recombinant human (rh) BMP-4 instead of the rhBMP-4 molecule.

Methods

The experimental composition of plasmid DNA rhBMP-4/PLG was prepared in Drs. Ozaki and Winn’s laboratory. We used the rat critical-sized calvaria defect (CSD) model that has been widely published to test bone regeneration. Five experimental groups consisted of: 1. Untreated CSD (negative control), 2. Poly(D,L-lactide-co-glycolide) (PLG), 3. Plasmid DNA rhBMP-4, 4. Plasmid DNA rhBMP-4/PLG, 5. Rat autologous bone graft (positive control). There were 10 rats (3-month-old male Long-Evans) per group. 10 replicates/treatment cohort will provide approximately 90% power (type I error = 5%) for detecting differences among treatments. Critical-sized defects were created using an 8-mm trephine. Rats were euthanized at 2-, 4-, and 8-weeks postsurgery. Quantitative data for bone formation included radiomorphometry and histomorphometry. Data were tested with correlative statistics consisting of linear regression analysis, ANOVA, and multiple comparisons analyses. Significance was established at p0.05.

Radiomorphometry: Radiographic images of each cranium were obtained. All radiographs were scanned and digitally stored at a high resolution, using computer-imaging software. A computerized radiodensity analysis was performed to quantitatively determine new bone formation in each specimen.

Histomorphometry: After radiography, all bone specimens were histologically and histomorphometrically analyzed. Sections were qualitatively analyzed for a description of cellular events and bone graft healing. Histomorphometry was performed to quantitatively evaluate features of new bone formation.

Results

Radiomorphometric: At week 2, little bone formation was seen in the rhBMP-4 plasmid DNA/PLG, untreated CSD, PLG alone, or rhBMP-4 plasmid DNA. By week 4, the mean average of the rhBMP-4 plasmid DNA/PLG was significantly greater than the untreated CSD, PLG alone, or the rhBMP-4 plasmid DNA (figure 1). The mean average of rhBMP-4 plasmid DNA/PLG was significantly lower than the mean radiopacity of the autograft group. At week 8, quantitative radiomorphometric data, continued to show the rhBMP-4 plasmid DNA/PLG had a significantly greater percent radiopacity than the untreated CSD, PLG alone, or the rhBMP-4 plasmid DNA. The mean average of the rhBMP-4 plasmid DNA/PLG group was significantly lower than the mean radiopacity of the autograft group (figure 3).

Histology and Histomorphometric: At week 2, little bone formation was observed histologically in the plasmid DNA rhBMP-4/PLG, untreated CSD, PLG alone, or rhBMP-4 plasmid DNA. A vast majority of the PLG was present in bright field and polarization microscopy. By week 4, minimal new bone formation was observed in the untreated CSD, PLG alone, or rhBMP-4 plasmid DNA groups, and fibrous connective tissue prevailed throughout (figure 2). By 4 weeks, the collagen matrix was apparently resorbed, while the porous PLG persisted. Defects receiving the rhBMP-4 plasmid DNA/PLG demonstrated many bony trabeculae and an abundance of new bone. Quantitative histomorphometric data showed the rhBMP-4 plasmid DNA/PLG had a significantly greater percent of new bone formation than the untreated CSD, PLG alone, or rhBMP-4 plasmid DNA. The autograft group exhibited a significantly greater mean bone formation than the rhBMP-4 plasmid DNA/PLG group. At 8 weeks, the new bone formed in the rhBMP-4 plasmid DNA/PLG treated wounds appeared more lamellar than at 4 weeks. There were no remnants of the collagen matrix, while the porous PLG persisted. As demonstrated by quantitative histomorphometry, the rhBMP-4 plasmid DNA/PLG group had a significantly greater percent of new bone formation than the untreated CSD, PLG alone, or rhBMP-4 plasmid DNA. The autograft group exhibited a significantly greater mean bone formation than the rhBMP-4 plasmid DNA/PLG group (figure 3).

Discussion

Recent advances in bioengineering and tissue engineering technology have broadened our knowledge and capacity to create tissues from engineered cells. Observations from the present study indicate that rhBMP-4 plasmid DNA combined with a PLG scaffold promoted significantly greater bone regeneration than an untreated CSD, defects containing PLG, or defects injected with the rhBMP-4 plasmid DNA. Furthermore, a relative low 50-µg dose of plasmid DNA promoted significant bone regeneration in a rat critical-sized calvarial defect. Future studies will apply this gene therapy construct to an alveolar cleft dog model and finally a non-human primate model. Although the challenges are significant, it is our intention to eventually reduce the clinical need for autogenous bone grafts to repair bony defects in children. If realized, this will lead to a reduction in surgical morbidity, hospital and surgical costs, and postoperative pain.

Figure 1: Radiographs of representative rat crania consisting of empty defect, carrier alone, plasmid alone, and plasmid/carrier groups. Each panel has a radiograph of 2-, 4-, and 8-week rat cranial defects. The plasmid/carrier group demonstrates new bone remineralization while the other treatment groups are generally radiolucent.

Figure 2: Panels depict CSDs after 4- and 8-week treatment intervals using Goldner trichrome stain. The plasmid/carrier group demonstrates new bone formation while the other treatment groups generally show fibrous tissue ingrowth.

Figure 3: Graphs representing radiomorphometry and histomorpometry for the defect, carrier alone, plasmid alone, plasmid/carrier and autograft groups.



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