¹Department of Clinical Oral Molecular Biology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan

²Department of Orthodontics, Faculty of Dental Science, Kyushu University, Fukuoka, Japan

Correspondence to: M Nakashima, Department of Clinical Oral Molecular Biology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan

The long-term goal of dental treatment is to preserve teeth and prolong their function. In dental caries an efficient method is to cap the exposed dental pulp and conserve the pulp tissue with reparative dentin. We examined whether growth/differentiation factor 11 (GDF11), a morphogen could enhance the healing potential of pulp tissue to induce differentiation of pulp stem cells into odontoblasts by electroporation-mediated gene delivery. Recombinant human GDF11 induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts, in mouse dental papilla mesenchyme in organ culture. The Gdf11 cDNA plasmid which was transferred into mesenchymal cells derived from mouse dental papilla by electroporation, induced the expression of Dsp. The in vivo transfer of Gdf11 by electroporation stimulated the reparative dentin formation during pulpal wound healing in canine teeth. These results provide the scientific basis and rationale for gene therapy for endodontic treatments in oral medicine and dentistry.

Gene Therapy 2002 9, 814-818. DOI: 10.1038/sj/gt/3301692

dental pulp; odontoblasts; regeneration; gene therapy; electroporation; growth/differentiation factor 11

The emerging fields of tissue engineering and regenerative medicine seek to replace or repair lost or damaged tissues due to disease, trauma and tumors.¹ Tissue engineering and regeneration requires three key ingredients: morphogenetic signals including growth and differentiation factors, responding stem cells and a scaffold of extracellular matrix.¹ In general, regeneration and repair recapitulates embryonic development. Bone morphogenetic proteins (BMPs) are multifunctional cytokines and widely distributed both in skeletal and non-skeletal tissues and have a major role in organogenesis. BMPs have actions beyond bone in neural, renal and cardiac development.^1,2 BMPs also play a role in differentiation of dentin^3,4,5,6,7,8 in teeth. The recent progress in molecular developmental biology permits the delivery of BMPs by gene therapy using optimal delivery.⁹ Growth/differentiation factor 11 (GDF11) is a novel member of the BMP/TGF family.^10,11,12 It was expressed in terminally differentiating odontoblasts,¹¹ implying a role in the differentiation of dental pulp stem cells into odontoblasts.¹¹ Therefore, we investigated whether the gene transfer of Gdf11 might stimulate odontoblast differentiation and reparative dentin formation in vitro and in vivo. The results revealed the potential utility of Gdf11 gene therapy in endodontic treatment in dentistry.

Viral vectors and non-viral techniques can be used for gene transfer in gene therapy. Although viral vectors provide gene transfer with high efficiency, attendant problems of cellular immunity due to adenoviruses or insertional mutagenesis due to retroviruses have been recognized.¹³ On the other hand, plasmid-mediated gene therapy, while minimizing immune responses, is inefficient. A potential method to overcome this dilemma is electroporation using pulsed electric fields to deliver DNA.^14,15,16,17 Many tissues respond to electroporation, and handling is relatively easy and rapid.¹⁸ Electroporation also has been used to deliver genes to living animals.^19,20,21 However, electroporation yields only a transient gene expression and not as efficient as viral vectors.¹⁸

In this investigation, we have optimized gene transfer of Gdf11 to pulp cells to initiate odontoblast differentiation in vitro and reparative dentin formation in vivo by electroporation for the endodontic treatment of pulp tissue regeneration and dentin repair. During terminal differentiation of odontoblasts, the expression of Gdf11 mRNA by in situ hybridization in mouse tooth germ¹¹ and by RT-PCR in the primary dental pulp cell culture has been demonstrated. The human recombinant GDF11 protein was used to explore the function of GDF11 in dental pulp cells. Differentiation of dentin-forming odontoblasts was monitored by three differentiation markers, dentin matrix protein1 (Dmp1), dentin sialoprotein (DSP) and osteocalcin. The expressions of these genes are known to increase during differentiation of pulp cells into odontoblast-like cells in pulp cell culture.^22,23,24 In murine developing molars, Dmp1 transcripts are expressed at the late bud stage, while Dsp mRNA is expressed later at the cap stage. Dmp1 expression is decreased in odontoblasts after the appearance of mineral, while Dsp exhibits sustained high expression.²⁵ In the primary pulp cell culture, Gdf11 was expressed initially on day 0 (not shown), day 5 and very weakly on day 9. Then it declined and disappeared on day 14. It reappeared with further differentiation on days 21 and 28 (Figure 1). The expressions of Dmp1 and Dsp decreased on day 9 and reappeared on day 21 in association with Gdf11 expression. Osteocalcin was expressed on day 28 (Figure 1), when mineralization was detected by Alizarin Red staining (data not shown). The expression pattern of Gdf11, Dmp1 and Dsp in vitro, together with the in situ expression results,¹¹ suggest that Gdf11 plays a role in the terminal differentiation of odontoblasts.

As Gdf11 was expressed during differentiation of pulp cells into odontoblasts, we next investigated the effect of beads soaked in recombinant human GDF11 on the response of mouse dental papilla mesenchyme derived from 17.5 days post coitum (dpc) tooth germ. The expression of Dsp mRNA was detected in the tissue surrounding the beads with GDF11 (Figure 2). On the other hand, as expected, no Dsp mRNA is seen around control BSA beads, suggesting that GDF11 stimulates differentiation of pulp stem cells into odontoblast lineage.

The current practice of endodontic treatment for pulp involves use of calcium hydroxide for pulp capping. It was of interest to utilize Gdf11 gene delivery for endodontic therapy. The plasmid, mouse Gdf11 driven in a pEGFP vector with TIMP promoter site was transfected by electroporation in primary pulp cell culture and in organ cultures of tooth germ (Figure 3). A high transfection efficiency was observed using green fluorescent protein (GFP) as marker for gene transfer. GFP was not detected in the culture with the pEGFP vector without electrotransfection and in the cultures without the pEGFP vector with electrotransfection (data not shown). A regimen of eight square-wave pulses were delivered at a frequency of 1 Hz, with a pulse length of 40 ms and 70 V in the multi-layered primary pulp cells, and with a pulse length of 10 ms and 100 V in the subconfluent primary pulp cells Figure 3a. In the organ cultures of mouse tooth germ, on the other hand, optimal delivery of Gdf11 plasmid was obtained by eight square-wave pulses delivered at a frequency of 1 Hz, with a pulse length of 10 ms and 10V Figure 3b. Seven days after electroporation, expression of Dsp mRNA was detected in the organ cultures transfected with Gdf11 (Figure 4d). The organ cultures transfected with pEGFP alone and those with pEGFP-Gdf11 without electrotransfection expressed no Dsp mRNA (data not shown).

The in vivo electroporation of Gdf11 in the amputated pulp of canine teeth has shown that the pulp cells differentiated into osteodentinoblasts and secreted osteodentin matrix around them and formed osteodentin 1 month after surgery (Figure 5a, c). In the control teeth transfected with pEGFP only, the differentiation of the pulp cells was not observed Figure 5b. Surgical amputation of pulp and the use of a capping agent, such as calcium hydroxide stimulates pulp cells to differentiate into odontoblasts and produce a small amount of dentin matrix. Some cells degenerate and leave a void in the spaces that they formerly occupied.²⁶ This kind of reparative dentin containing few or no dentinal tubules is called osteodentin,^27,28,29 distinguishing from tubular dentin. After application of morphogens, such as recombinant human BMP2 and BMP4 proteins with collagen matrix,⁵ stimulation of a large amount of osteodentin formation was observed. The present result of GDF11 gene therapy in pulp has demonstrated osteodentin matrix formation as observed with BMP protein therapy.⁵ The osteodentin matrix production is a biological response to repair induced by Gdf11 gene therapy. Immediately adjacent to the electrode there was a lake of erythrocytes in a plasma clot possibly due to thermal effects Figure 5d. It is possible to avoid the thermal effects of electroporation by the use of ultrasound-mediated gene delivery³⁰ during endodontic treatment. In conclusion, transfer of Gdf11 gene by electroporation to pulp cells in an amputated tooth in vivo induced new reparative dentin. We are aware of the important implications for endodontic treatment by gene therapy in dentistry.

Table 1.

Acknowledgements

The authors are grateful to K Hirakawa and T Yamauchi of Tokiwa Science for their help and to Genetic Institutes for their kind gift of recombinant human GDF11. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, No. 11470406.

1 Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nature Biotech 1998; 16: 247-252.

2 Kirker-Head CA. Potential applications and deliver strategies for bone morphogenetic proteins. Adv Drug Del Rev 2000; 43: 65-92.

3 Nakashima M. The induction of reparative dentine in the amputated dental pulp of the dog by bone morphogenetic protein. Archs Oral Biol 1990; 35: 493-497.

4 Nakashima M. Induction of dentin formation on canine amputated pulp by recombinant human bone morphogenetic protein (BMP)-2 and -4. J Dent Res 1994; 73: 1515-1522. MEDLINE

5 Nakashima M. Induction of dentine in amputated pulp of the dogs by recombinant human bone morphogenetic proteins-2 and -4 with collagen matrix. Archs Oral Biol 1994; 39: 1085-1089.

6 Rutherford RBet al. . Induction of reparative dentin formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 1993; 38: 571-576. MEDLINE

7 Rutherford RBet al. . Time course of the induction of reparative dentin formation in monkeys by recombinant human osteogenic protein-1. Arch Oral Biol 1994; 39: 833-838. MEDLINE

8 Rutherford RB, Spangberg L, Tucker M, Charette M. Transdentinal stimulation of reparative dentine formation by osteogenic protein-1 in monkeys. Arch Oral Biol 1995; 40: 681-683. MEDLINE

9 Winn SR, Hu Y, Sfeir C, Hollinger JO. Gene therapy approaches for modulating bone regeneration. Adv Drug Del Rev 2000; 42: 121-138.

10 McPherron AC, Lawler AM, Lee SJ. Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11. Nature Genet 1999; 22: 260-264. Article MEDLINE

11 Nakashima M, Toyono T, Akamine A, Joyner A. Expression of growth/differentiation factor 11, a new member of the BMP/TGF superfamily during mouse embryogenesis. Mech Dev 1999; 80: 185-189. Article MEDLINE

12 Gamer LWet al. . A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos. Dev Biol 1999; 208: 222-232. Article MEDLINE

13 Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995; 270: 404-410. MEDLINE

14 Neumann EM, Schaefer-Ridder YW, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982; 1: 841-845. MEDLINE

15 Fromm M, Taylor LP, Walbot V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 1985; 82: 5824-5828. MEDLINE

16 Andreason GL, Evans GA. Introduction and expression of DNA molecules in eukaryotic cells by electroporation. Biotechniques 1988; 6: 650-660. MEDLINE

17 Chowrira GM, Akella V, Fuerst PE, Lurquin PF. Transgenic grain legumes obtained by in planta electroporation-mediated gene transfer. Mol Biotechnol 1996; 5: 85-96. MEDLINE

18 Muramatsu T, Nakamura A, Park HM. In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals. Int J Mol Med 1998; 1: 55-62. MEDLINE

19 Weaver JC. Electroporation: a general phenomenon for manipurating cells and tissues. J Cell Biochem 1993; 51: 426-435. MEDLINE

20 Hofmann GA. Instrumentation. Nickoloff JA (eds); Methods in Molecular Biology. Electroporation Protocols for Microorganisms Humana Press, 1995, pp 27-45.

21 Dev SB, Hofmann GA. Electrochemotherapy - a novel method of cancer treatment. Cancer Treat Rev 1994; 20: 105-115. MEDLINE

22 Nakashima M, Nagasawa H, Yamada Y, Reddi AH. Regulatory role of transforming growth factor-, bone morphogenetic protein-2, and protein-4 on gene expression of extracellular matrix proteins and differentiation of dental pulp cells. Dev Biol 1994; 162: 18-28. Article MEDLINE

23 Yokose Set al. . Establishment and characterization of a culture system for enzymatically released rat dental pulp cells. Calcif Tissue Int 2000; 66: 139-144. Article MEDLINE

24 Couble MLet al. . Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif Tissue Int 2000; 66: 129-138. Article MEDLINE

25 D'Souza RNet al. . Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Min Res 1997; 12: 2040-2049.

26 Avery JK. Dentin. Bhaskar SN (eds); Orban's Oral Histology and Embryology Mosby, 1980, pp 107-140.

27 Baume LJ. Dental pulp conditions in relation to carious lesions. Int Dent J 1970; 20: 309-337. MEDLINE

28 Takuma S, Yanagisawa T, Lin WL. Ultrastructural and microanalytical aspects of developing osteodentin in rat incisors. Calcif Tissue Res 1977; 24: 215-222. MEDLINE

29 Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent 2000; 28: 77-92. MEDLINE

30 Newman CM, Lawrie A, Brisken AF. Cumberland DC. Ultrasound gene therapy: on the road from concept to reality. Echocardiography 2001; 18: 339-347. MEDLINE

31 Nakashima M. Establishment of primary cultures of pulp cells from bovine permanent incisors. Archs Oral Biol 1991; 36: 655-663.

32 Gu Ket al. . Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci 2000; 108: 35-42. Article MEDLINE

33 Kiefer MC, Saphire AC, Bauer DM, Barr PJ. The cDNA and derived amino acid sequences of human and bovine bone Gla protein. Nucleic Acids Res 1990; 18: 1909. MEDLINE

34 Alonso S, Minty A, Bourlet Y, Buckingham M. Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol Evol 1986; 23: 11-22. MEDLINE

35 Platt KA, Michaud J, Joyner AL. Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals a conservation of pathways between flies and mice. Mech Dev 1997; 62: 121-135. Article MEDLINE

Figure 1 RT-PCR analyses for Gdf11 (product size: 0.5 kb), Dmp1 (0.5 kb), Dsp (0.4 kb), Osteocalcin (0.3 kb) and -actin (0.5 kb) in the primary dental pulp cell culture. The bovine dental pulp cells were isolated³¹ and inoculated at a density of 1 ´ 10⁵ cells/ml, and 5, 9, 14, 21 and 28 days after cultivation, total RNA were isolated using Trizol (Life Technologies, Rockville, MD, USA). First-strand cDNA syntheses were performed by reverse transcription using the SuperScript preamplification system (Life Technologies). The design of the oligonucleotide primers was based on published cDNA sequences: Gdf11,¹¹ Dmp1 (Hirst KL, Ibaraki K, Young MF, Dixon MJ. GenBank accession number U47636 (unpublished)), Dsp,³² Osteocalcin³³ and -actin³⁴ (Table 1). PCR amplifications were performed for 35 cycles (94°C for 30 s, 65°C for 1 min, 72°C for 1 min) for Gdf11, 35 cycles (94°C for 30 s, 60°C for 1 min, 72°C for 1 min) for Dmp1, 36 cycles (94°C for 30 s, 62°C for 1 min, 72°C for 1 min) for DSP, 34 cycles (94°C for 30 s, 55°C for 1 min, 72°C for 1 min) for osteocalcin, and 34 cycles (94°C for 30 s, 55°C for 1 min, 72°C for 1 min) for -actin. The PCR conditions used were optimized and standardized. Note the expressions of Dmp1 and Dsp, differentiation markers for dentin-forming odontoblasts was associated with reappearance of Gdf11 on day 21. The mineralization was confirmed by Alizarin Red staining. The experiment was repeated three times and one representative experiment is presented.

Figure 2 Whole-mount RNA in situ hybridization analysis showing the induction of the expression of Dsp mRNA by recombinant human GDF11 in vitro organ culture. The dental papillae were isolated from enamel epithelium and odontoblastic layer in tooth germ at 17.5 dpc from ICR mouse (CREA, Japan) and cultured on a Nucleopore Track-Etch membrane (Whatman, Springfield Mill, UK) supported by metal grid in Fitton-Jackson modified BGJb medium (Life Technologies) supplemented with 2% bovine calf serum (JRH Biosciences, Lenexa, KS, USA) and 50 g/ml L-ascorbic acid phosphate (Wako Pure Chemical Industries, Osaka, Japan). Agarose beads of 5 l of Affi-Gel Blue Gel (BioRad Laboratories, Hercules, CA, USA) was soaked with 50 g of recombinant GDF11 (kindly provided by Genetics Institute, Cambridge, MA, USA) (a), or 50 g of bovine serum albumin (Life Technologies) (b) overnight at 4°C. The beads were placed on the dental papillae tissue in the organ culture for 7 days. In situ hybridization was performed using mouse DIG DSP probe as described previously.³⁵ For the mouse probe, the 1080 bp clone (9586-10665) was obtained by PCR from mouse genomic DNA (Clontech, Palo Alto, CA, USA) using primers, DSP-5'-3 (5'-CGCGAATTCGACAGGAGAGATGTGCAGACT-3') and DSP-3'-4 (5'-TACGGATCCAGGAGGTGAGCACCTGAGAA-3'), subcloned into pBlueScript II SK(-), linearized with HindIII and transcribed by T3 polymerase for the antisense probe. The experiment was repeated five times and one representative experiment is presented.

Figure 3 The subconfluent primary pulp cells (a) and the tooth germ (b) showing the high efficiency of electrotransfection by the pEGFP-N3 (Clontech), 1 day after cultivation. Eight square-wave pulses at a frequency of 1 Hz, with a pulse length of 10 ms, and 100 V (a) or 10 V (b). The optimal conditions for electrotransfection were examined in the subconfluent primary pulp cells using a comb-type electrode with a 5 mm distance (gap) between stainless steel wires, 30 mm in diameter, using a electroporator, EDIT-TYPE CUY21 (Tokiwa Science, Fukuoka, Japan). The optimal conditions were determined by counting the number of GFP particles, under a fluorescent dissection microscope (Leica, Heerbrugg, Switzerland) with GFP fluorescent filter at 488 nm. For the electrotransfection in the organ culture, a stainless steel electrode needle with a 1 mm gap (Tokiwa Science) was placed on the surface of the tissue.

Figure 4 Whole-mount RNA in situ hybridization analysis showing the induction of the expression of Dsp mRNA, 7 days after electrotransfection with Gdf11 in vitro. The mouse dental papillae mesenchyme transfected with the pEGFP vector with TIMP promoter (a, c) or with the mouse Gdf11 driven by TIMP promoter-pEGFP vector (b, d). The plasmid pEGFPN3-TIMP-mGdf11 was constructed by ligating the 140 bp PCR product of TIMP and the 1.0 kb PCR product of mouse Gdf11.¹¹ The plasmid was confirmed by sequence. The purification was performed by Wizard Plus Midi Prep (Promega, Madison, WI, USA) and the plasmids were adjusted to a concentration of 20 g/l in phosphate buffer saline. The dental papillae were isolated in the same manner as described in Figure 2, and 0.5 l of the ice-cold plasmid was applied on the dental papillae. The condition of the electrotransfection was eight square-wave pulses at a frequency of 1 Hz, with a pulse length of 10 ms and 10 V. Note the much stronger expression of Dsp mRNA in the mesenchyme electrotransfected with Gdf11 (d) than that with GFP only (c). The experiment was repeated three times and one representative experiment is presented.

Figure 5 The in vivo electroporation of Gdf11 in the amputated pulp of dogs. Tissues were examined after 1 month. A total of 20 teeth from five young adult dogs weighting 15-18 kg were used. Surgical anesthesia was obtained by intravenous administration of 35 mg pentobarbital sodium per kg body weight. The canine teeth were treated, and an exposure was made using a diamond round burr at high speed. After washing with a 5% solution of sodium hypochlorite and a 3% solution of H₂O₂, the amputation was carried out by means of a round metal burr at low speed. Twenty micrograms of pEGFP/Gdf11 plasmid (a, c, d) and pEGFP plasmid as a control (b) in phosphate buffer saline was applied by electroporation in the amputated pulp tissue in a similar condition as described above; 10 square-wave pulses were delivered at a frequency of 1 Hz, with a pulse length of 1 ms and 10 V. The cavity was filled with zinc phosphate cement and composite resin. Note the formation of the thick osteodentin matrix (OD) (a, c) beneath the amputated site (arrows) in response to Gdf11 gene therapy. The newly induced reparative dentin with intercellular matrix contains only few dentinal tubules (c). The proliferation of pulp tissue in the cavity above the amputated site (a, b). No osteodentin matrix formation in the control (b). Pulp tissue (Pu). The plasma clot (Cl) immediately adjacent to the electrode (d).

Table1 Primers used for RT-PCR amplification on Gdf11 and differentiation markers for odontoblasts