Hard tissue formation on dentin surfaces applied with recombinant human bone morphogenetic protein-2 in the connective tissue of the palate
ABSTRACT The purpose of this study was to evaluate whether hard tissue might be formed on dentin surfaces applied with recombinant human bone morphogenetic protein-2 (rhBMP-2) in palatal connective tissue. Fifty-eight dentin blocks were prepared from rat roots, demineralized with 24% EDTA (pH 7.0), applied with 0, 50 and 100 microgram/ml rhBMP-2, and labeled as groups 0, 50 and 100. The dentin blocks were then transplanted into palatal connective tissue of rats, and specimens were prepared at two and four weeks after surgery for histologic and histomorphometric examinations. The results showed that the percentage of newly formed hard tissue in relation to the total dentin block surface length in groups 0, 50 and 100 was 0.0%, 2.8% and 4.4% at two weeks, and 0.0%, 1.6% and 12.8% at four weeks, respectively. New hard tissue formation in groups 50 and 100 was significantly promoted as compared to group 0 (p < 0.01). These findings thus indicate that rhBMP-2 application to dentin enhanced new hard tissue formation on dentin surfaces in the connective tissue of the palate.
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ABSTRACT: Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat beta-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 x 10(6) Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.Gene Therapy 02/2005; 12(1):75-86. DOI:10.1038/sj.gt.3302386 · 4.20 Impact Factor
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ABSTRACT: Recent studies have shown that bone morphogenetic protein-2 (BMP-2) stimulates mineralization and osteoclast differentiation. Osteoclastic resorption by BMP-2 application may play an important role in the regulation of new cementum-like tissue formation on the dentin surfaces. Therefore, this study aimed to examine the effect of BMP-2 application on dentin resorption and cementum-like tissue formation at the dentin surfaces. Seventy-two flat dentin blocks were prepared from rat roots and treated with 24% EDTA. Each block was assigned to group 0, group 100, or group 400, and immersed correspondingly in 0, 100, or 400 microg/ml BMP-2. The dentin blocks were then implanted into palatal connective tissue of rats, and specimens were prepared 2, 4 and 8 wk after surgery for histologic and histomorphometric analyses. BMP-2 caused a dose-dependent increase in dentin resorption by osteoclastic cells. New cementum-like tissue was randomly formed on parts of the nonresorbed and resorbed dentin surfaces in groups 100 and 400. Dentin resorption in groups 100 and 400 was significantly greater than group 0 (p < 0.01). However, at 8 wk, new cementum-like tissue formed in 41.8% of group 100, as compared with 16.2% of group 400 (p < 0.05). Dentin resorption was stimulated by a high dose of BMP-2, and cementum-like tissue was induced by a low dose of BMP-2, effectively suggesting that BMP-2 application, at an appropriate dose, to a dentin surface may enhance periodontal regeneration.Journal of Periodontal Research 09/2006; 41(4):311-5. DOI:10.1111/j.1600-0765.2006.00878.x · 2.22 Impact Factor
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ABSTRACT: Palatoplasty has the undesired side effect of impaired mid-facial growth. To avoid this problem, we propose an alternative to palatoplasty. We hypothesize that if BMP-2 is injected together with a carrier into the periosteum of the cleft palate borders, border volume will increase and connective tissue cells will be activated to produce extra bone. Once these borders supported by bone reach the midline, extraction of their covering epithelia with trypsin will permit adhesion of the underlying tissues. We investigated in vitro the ability of cleft palate connective tissue cells to produce extra bone in the presence of BMP-2 and the possibility of using trypsin to remove the epithelium covering the cleft palate borders without impairing the underlying tissues' ability to adhere. We used the cleft palate presented by tgf-beta(3) null mice and small fragments of human cleft palate mucoperiosteum as models. Immunolabeling BMP-2-treated or untreated cultures with TUNEL and anti-osteocalcin or PCNA antibodies was performed. The epithelium of the cleft palate borders was removed with a trypsin solution, and the de-epithelialized tissues were cultured in apposition. BMP-2 induces differentiation toward bone on cleft palate connective tissue cells without producing cell death or proliferation. Trypsin removal of the cleft palate margins' epithelium does not impair the underlying tissues' adhesion. It is possible to generate extra bone at the cleft palate margins and to chemically eliminate their covering epithelia without damaging the underlying tissues, which allows further investigation in vivo of this new approach for cleft palate closure.Journal of Surgical Research 04/2007; 138(1):111-20. DOI:10.1016/j.jss.2006.07.033 · 2.12 Impact Factor