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Targeting and Immobilization of Bioactive Peptides on Dentin Matrix
1 Department of Endodontics and Correspondence: * corresponding author, yumigimai{at}comcast.net, jscsong{at}yahoo.com
The regeneration of structurally/functionally competent tooth root cementum is a critical step for the successful restoration of periodontal attachment. In this study, we tested whether a poly-glutamic acid-rich domain and glutamine-containing transglutaminase substrate can be used to target biologically active peptides to the mineralized root matrix and to bind such peptides covalently to the organic matrix. As a biologically active model molecule, the integrin-binding motif, RGD, was used. The effects of immobilization of such synthetic peptides to the dentin matrix on cementoblastic adhesion in vitro and cementogenesis in vivo were studied. In vitro, cementoblastic adhesion improved significantly when the dentin surface contained covalently bound peptides. In vivo, this bound peptide significantly increased cementum formation compared with that attained in control conditions. Transglutaminase-catalyzed covalent binding of bioactive peptides targeted to mineralized collagenous dentin matrix via the poly-glutamate domain can be readily achieved. This approach offers potential for clinical use in periodontal regeneration.
Key Words: peptide • dentin • bioactive • periodontal • regeneration
The regeneration of cementum on previously exposed root surfaces is an important step in periodontal regeneration (Grzesik and Narayanan,2002). It is apparent that targeted delivery/sequestration of bioactive substances onto the tooth root surface should enhance this process. Furthermore, for such substances to influence cementogenesis, a relatively late stage in the healing process, it is imperative that they remain bound to the root for a considerably long time. Bone sialoprotein (BSP), a major non-collagenous protein found in cementum matrix, is believed to bind hydroxyapatite via several domains of 8 contiguous glutamic acid residues (Somerman et al., 1990; MacNeil et al., 1995; Fujisawa et al., 1996; Saygin et al., 2000).
Transglutaminases (TGase) are enzymes that catalyze the rapid generation of covalent crosslinks between proteins during important biological processes, producing a supramolecular structure with extra rigidity and resistance against proteolytic degradation (Lorand and Graham, 2003). Transglutaminases crosslink with proteins through an acyl-transfer reaction between the carboxamide group of peptide-bound glutamine residue (acyl donor) and the amino group of peptide-bound lysine residue (acceptor), resulting in a We hypothesized that peptides containing a poly-glutamic acid domain can be utilized to target bioactive molecules to mineralized tooth matrix. Also, by including the transglutaminase substrate domain, glutamine residues, within the peptide and simultaneously supplying exogenous transglutaminase, we could theoretically achieve a stable binding of the peptide to collagen exposed on the tooth root surface. Thus, such a system should, in principle, enhance the regenerative process. For technical reasons (i.e., the stability and the relative ease of observing the biological effects), and based on the current knowledge of the critical role of integrins for mesenchymal cell survival, proliferation, and differentiation, we chose the integrin-binding RGD motif as a model of a bioactive molecule for this study. In addition, several RGD-containing proteins (BSP, cementum attachment protein, and collagen) are present in native cementum (Hynes, 1992; Ganss et al., 1999; Ivanovski et al., 1999; Nanci, 2003), suggesting that they may have anabolic effects on cemento and osteoprecursors via integrin-mediated/modulated pathways (Nakae et al., 1991; MacNeil and Somerman, 1993, 1999; Narayanan and Bartold, 1996). Therefore, it was reasonable to hypothesize that the interaction of committed cementoblastic cells with immobilized integrin ligand would result in increased cell adhesion in vitro, which, very likely, would enhance cementogenesis in vivo.
All the relevant protocols were approved by the appropriate institutional committees (IRB, Institutional Animal Care Committee [IACUC]) at the University of Pennsylvania. Informed consent was obtained for the use of human tissues.
Cell Culture
Dentin Substrates Tooth root dentin slices, 1 mm thick, were prepared with the use of a circular diamond saw (Buehler, Lake Bluff, IL, USA). Four or 8 small chambers, 1 mm wide and 0.5 mm deep, were prepared on each slice by means of a round carbide bur (#7002, Dentsply, York, PA, USA). All dentin substrates used were rehydrated in PBS for 30 min before use.
Peptides
The peptide and transglutaminase (Sigma, St. Louis, MO, USA) mix was prepared ex tempore for all of the following experiments, when applicable.
Evaluation of Targeting and Immobilization of Peptides
Cell Attachment Assays In another set of experiments, transglutaminase and either fully functional peptide, TG-null peptide, or MB-null peptide were applied to root dentin slices. The stained cells within the chamber were counted with an inverted microscope (100x magnification), and the obtained data were subjected to statistical analysis by ANOVA.
In vivo Cementogenesis Assay
The data were analyzed by non-paired Student’s t test. At least 3 different transplants/group were analyzed.
Evaluation of Targeting and Immobilization of Peptides The results from the evaluation of poly-glutamate-mediated targeting and transglutaminase-catalyzed crosslinking of biotinylated synthetic peptide to mineralized tissue are shown in Fig. 1  . The greatest intensity of staining was observed within the dentin chambers that were treated with both the peptide and transglutaminase, followed by those treated with peptide alone and transglutaminase alone. These results indicate that transglutaminase-mediated crosslinking of peptides (peptide and transglutaminase group) resulted in significantly higher (about three-fold) retention of synthetic peptide compared with the peptide bound via the poly-glutamate alone (peptide alone group) (Fig. 1).
Cell Attachment Assays Significantly greater numbers of HCDC were attached to the dentin matrix surfaces that were treated with the fully functional peptide (TG-MB-RGD) and transglutaminase compared with the dentin surfaces that were treated with transglutaminase alone or PBS alone. Also, attached cells spread better on dentin matrix surfaces treated with the fully functional peptide in the presence of transglutaminase than in other conditions (Fig. 2a ), indicating that the transglutaminase-catalyzed crosslinking of peptides to mineralized human dentin matrix promoted enhanced cell adhesion.
Further, when quantifying the cell adhesion as a function of peptide composition, we found that the highest number of cells was attached to the dentin matrix surface that was treated with the fully functional peptide (P < 0.05 compared with control), followed by the TG-null peptide and MB-null peptide (although the latter tendency did not reach statistical significance) (Fig. 2b ). There was no significant difference in cell attachment between the modified peptides and transglutaminase groups when applied alone and the PBS alone group (not shown). These results show that, to achieve maximum biological effect, all of the active components (i.e., both TG and MB domains) in the synthetic peptide are required for efficient binding to the dentin matrix.
In vivo Cementogenesis Assay We evaluated the efficiency of cell loading of transplants by extracting and measuring DNA content from cells within prepared transplants. We found that the loading procedure was highly efficient, and that 96% of the initially applied cells were retained in the transplants at the end of the procedure. Further, the modifications of the vehicles (or the type of vehicle) had no effect on the number of cells retained.
In vivo, the newly formed cementum was tightly bound to the dentin matrix substrates pre-treated with fully functional peptide and transglutaminase (Fig. 3a
Bioactive molecules such as growth factors, extracellular matrix components, and cytokines are involved in cementogenesis (Nakae et al., 1991; MacNeil and Somerman, 1993, 1999; Pitaru et al., 1994; Narayanan and Bartold, 1996). In this study, we developed a new strategy to target and stably incorporate bioactive molecules into the mineralized matrix of the tooth root. This strategy is based upon the binding of poly-glutamic acid residues to the surface of mineralized matrix, followed by the transglutaminase-catalyzed immobilization of these peptides to the organic part of the root matrix. The effectiveness of this strategy was confirmed in vitro. Using this approach, we have demonstrated a significant increase in the amount of peptide bound to dentin matrix. In addition, when the RGD cell-binding motif was included, a significant increase in cementoblastic cell attachment was evident in vitro compared with that in controls. Last, the combination of these bioactive treatments enhanced cementogenesis in vivo, as shown by an increase in deposition of mineralized cementum-like tissue in an in vivo transplantation assay. Tooth root dentin modified with mutated functional peptides and transglutaminase did not show enhanced cementogenesis over control, unmodified tooth root dentin. These dramatic differences likely result from the lack of initial targeting/sequestration of the peptide (MB-null peptide) to the dentin surface. Subsequently, without covalent binding (crosslinking), the peptide (TG-null peptide) was not biologically active, resulting in reduced cell attachment. Our studies demonstrated a significant biological advantage associated with the immobilization of mineralized dentin matrix. Although further studies will be required, it is likely that our approach promotes site-specific delivery/sequestration and increased density of the material to the confined area of interest (mineralized matrix). We speculate that by using this approach, we not only induce and enhance cementogenesis, but we can also prevent overgrowth of induced tissue, sometimes seen as ankylosis (Selvig et al., 2002). Another advantage is the elimination of a carrier or delivery vehicle for the bioactive materials, which, in some situations, may hinder the intimate integration of the newly formed tissue with the tooth root surface (Bosshardt et al., 2005). Our in vivo results may also help explain why adsorption without subsequent crosslinking of whole adhesive proteins onto tooth root surfaces did not yield consistent enhancement of periodontal regeneration in vivo (Trombelli et al., 1996; Kurtis et al., 2002). Based on our data, it appears that the stable binding of bioactive adhesive molecules is essential. These in vivo experiments show that, if peptides are not covalently bound, there is either no effect or a decrease in the amount of cementum formed. In addition, when peptides are not stably bound, the separation of newly formed cementum from the underlying carrier matrix becomes more evident. It can be suggested that this phenomenon results, in part, from potential leakage of peptide that, in turn, may act as an integrin antagonist (Grzesik and Robey, 1994). As a result, both the cementum formation and the cementum-dentin interface are compromised. Thus, stable binding of the bioactive molecule to the root surface is critical for cementum regeneration to occur. The cellular and molecular interactions required for cementum formation are not well-elucidated. However, using a modified substrate and committed cementogenic cells, we observed enhanced cementogenesis. Further, no other cell types residing in the periodontium were present in our experimental system, and, for the in vivo experiments, no immune response was present. It is also likely that less-differentiated cementogenic precursor cells and perhaps even the non-committed stem-cell-like cells would possibly respond similarly to the fully committed cementoblastic cells; however, the technology for either the isolation of early cementoblastic precursors/progenitors or cementum-inducing factors is lacking at this time. Thus, it is not possible to design studies addressing these issues experimentally. In conclusion, this study demonstrates that bioactive peptides can be engineered to target mineralized tissue via mineral-binding poly-glutamic acid residues, and that these can be efficiently bound to tooth root matrices by transglutaminase-catalyzed crosslinking. In principle, this method can be used to target and stably bind any bioactive molecule to a mineralized matrix.
We thank Dr. Edward Macarak for critical review of the manuscript. This investigation was supported by National Institutes of Health/National Institute of Dental and Craniofacial Research grants DE-13475 and DE-14600.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication June 21, 2006. Revision received May 6, 2007. Accepted for publication June 7, 2007.
Journal of Dental Research, Vol. 86, No. 10,
968-973 (2007)
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ABSTRACT
INTRODUCTION
(
-glutamyl) lysine isopeptide bond (
-MEM medium (SFM) at a density of 1 x 105/mL. A 1-µL quantity of medium containing the cells was then added to each dentin chamber. Dentin slices were then incubated at 37°C for 24 hrs, washed with PBS, fixed in 4% formaldehyde, and then stained with0.25% Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, CA, USA) so that attached cells could be visualized. Images of cell attachment to the dentin substrates were captured with an inverted microscope (Nikon Diaphot 300) equipped with a Nikon Digital Still Camera DXM 1200 (Nikon Instrument Inc., Melville, NY, USA) and connected to an IBM-compatible PC computer equipped with Nikon ACT-1 Software (Nikon Instrument Inc.). 
