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RESEARCH REPORT |
1 Craniofacial and Skeletal Diseases Branch,
2 Clinical Research Core, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Building 30, Room 222, NIDCR/NIH, 30 Convent Drive MSC-4320, Bethesda, MD 20892;
3 Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Frome Road Adelaide 5000, South Australia, Australia;
*corresponding author, sshi{at}dir.nidcr.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: bone marrow stromal stem cell • dental pulp stem cell • osteoblast • odontoblast • transplantation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Transplantation
Approximately 4.0 x 106 of ex vivo expanded BMSSCs and DPSCs were mixed with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc., Warsaw, IN, USA) and then incubated at 37°C for 2 hrs. After centrifugation at 1500 rpm for 7 min, the supernatant was removed. The cell pellets with HA/TCP were transplanted into 10-week-old immunocompromised beige mice (NIH-bg-nu-xid, Harlan Sprague-Dawley, Indianapolis, IN, USA) as previously described (Krebsbach et al., 1997; Gronthos et al., 2000). Midlongitudinal skin incisions were made on the dorsal surface of each mouse, and subcutaneous pockets were made by blunt dissection. A single transplant was placed in each pocket, with up to 4 transplants per animal. These procedures were performed in accordance with specifications of an NIDCR/NIH IRB-approved animal protocol (NIDCR #02-222). The transplants were recovered at 2, 4, 8, and 16 wks post-transplantation, fixed with 4% formalin, decalcified with buffered 10% EDTA (pH 8.0), and then embedded in paraffin. For DPSC/dentin transplantation, the roots of the third molars were cut to expose the pulp chamber, a thin layer of pulpal dentin surface was removed by means of a carbide bur, and the exposed surface was treated with 1% acetic acid for 10 min at room temperature and then washed three times with PBS. Approximately 2.0 x 106 DPSCs were loaded onto the acid-treated/PBS-washed dentin surface and incubated under the cell culture medium at 37°C for 12 hrs. The root foramen was sealed with Gelfoam (absorbable gelatin sponge, Pharmacia & Upjohn Company, Kalamazoo, MI, USA), and the culture medium was removed before the transplantation. The DPSC/dentin complexes were then transplanted subcutaneously. Acid-treated dentin samples cultured with BMSSC (2.0 x 106) were transplanted as controls.
In situ Hybridization
A human-specific alu probe labeled with digoxigenin was used for in situ hybridization. The probe was prepared by PCR containing 1x PCR buffer (Perkin Elmer, Foster City, CA, USA), 0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP, 0.065mM dTTP, 0.035 mM digoxigenin-11-dUTP, 10 pmol of specific primers, and 100 ng of human genomic DNA as template. Primers for alu (GenBank Accession Number X53550) included sense, 5'-TGGCTCACGCCTGTAATCC-3' (base number 90-108), and antisense, 5'-TTTTTTGAGACGGAGTCTCGC-3' (base number 344-364). Unstained sections were deparaffinized and hybridized with the digoxigenin-labeled alu probe by means of the mRNAlocator-Hyb Kit (Cat. #1800; Ambion, Inc., Austin, TX, USA).
Immunohistochemistry
The BMSSC and DPSC transplants were fixed, decalcified, and embedded in paraffin, and 5-micron tissue sections were incubated with the primary antibodies at room temperature for 1 hr. Primary antibodies used were against: bFGF, MMP-9 (1:200 dilution; rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); mitochondria (1:100 dilution; rabbit anti-human-specific, Chemicon, Temecula, CA, USA); and dentin sialoprotein (1:200 dilution of LF-151) (Gronthos et al., 2002). Histostain SP Kits were used for biotinylated second antibodies and HRP enzyme-avidin conjugate incubation according to the manufacturer’s instructions (Zymed Laboratories Inc., South San Francisco, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Only approximately 20-30% of the transplants (2 out of 9 for BMSSC and 3 out of 10 for DPSC) showed a minimal amount of bone or dentin formation (Figs. 1a, 1e). At 4 wks post-transplantation, BMSSCs and DPSCs differentiated into osteoblasts and odontoblasts and generated bone and dentin, respectively, on the surface of the HA/TCP in all transplants (Figs. 1B, 1F). At 8 wks post-transplantation, BMSSCs generated a bone/marrow organ structure in which abundant bone tissue was formed and hematopoietic marrow elements replaced the connective tissue (Fig. 1C). In contrast, DPSCs generated a dentin/pulp-like complex where the pulp-like tissue was comprised of fibrous connective tissue, blood vessels, and odontoblasts associated with newly formed dentin (Fig. 1G). Moreover, transplanted BMSSCs and DPSCs continued to generate bone and dentin at least until 16 wks post-transplantation, at which time they formed a mature bone/marrow organ structure and dentin/pulp complex, respectively (Figs. 1D, 1H). The transplanted human BMSSCs and DSPCs were found either attached to the surface of the HA/TCP carrier or in the connective tissue compartment 2 wks post-transplantation (Figs. 2A, 2C). Most of the surviving BMSSCs differentiated into either osteoblasts or osteocytes, and the number of BMSSCs present in the connective tissue compartment decreased with time (Fig. 2B). However, DPSCs not only differentiated into odontoblasts that remained directly associated with the contiguous transplant carrier HA-dentin matrix, but also continued to reside in the connective tissue compartment even 8 wks post-transplantation (Fig. 2D).
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). Following bone formation at 4 wks post-transplantation, the number of cells expressing bFGF was significantly higher (Fig. 3B). This high level of bFGF expression continued until the connective tissue was replaced by hematopoietic marrow elements at 8 wks post-transplantation, at which point the level of bFGF expression was significantly diminished (Figs. 3C, 3D). There was no bFGF expression in DPSC transplants at any time point, and therefore bFGF appeared not to play an important role in the formation of the dentin/pulp-like complex (Figs. 3G, 3H).
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); however, it was highly expressed in the connective compartment of BMSSC transplants after 4 wks (Fig. 3F), and this elevated expression significantly diminished with the formation of hematopoietic marrow elements (data not shown). Like bFGF, MMP-9 was not seen in the dentin/pulp complex of the DPSC transplants. VEGF was found to be equally expressed in BMSSC and DPSC transplants, along with newly formed blood vessels (Figs. 3I, 3J).
Dentinogenesis of DPSCs
To assess the potential contribution of DSP to dentinogenesis, we examined the expression of DSP in dentinogenic cells of DPSC transplants and osteogenic cells of BMSSC transplants. Dentinogenic cells, similar to odontoblasts in human dental pulp, clearly showed a positive immunostaining for DSP antibody (Figs. 4A, 4C). DSP was not detectable in osteogenic cells of human bone and BMSSC transplants by immunohistochemical staining (Figs. 4B, 4D).
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). In contrast, DPSCs were capable of generating a reparative dentin-like structure directly on the surface of human dentin (Fig. 4F). Like reparative dentin formation in the human pulp-dentin system, the newly formed dentin did not contain an organized dentinal tubule structure, a result different from that shown in DPSC/HA/TCP transplants (Fig. 1G). The reparative dentin-like structure was initiated on the acid-treated human dentin surface that provided a scaffold for the dentinogenesis of DPSCs. Newly formed tissue was generated by dentinogenic cells located on its surface and cells trapped inside the tissue (Fig. 4F). To characterize the newly regenerated reparative dentin-like tissue on the pre-existing human dentin surface, we used immunohistochemical staining to show that the pre-existing dentin scaffold and the dentinogenic cells responsible for forming reparative dentin-like tissue were positive for DSP antibody staining (Fig. 4G). Connective tissue failed to show immunopositive staining for DSP antibody (Figs. 4G, 4H). Interestingly, only the peritubular dentin structure of the pre-existing dentin scaffold was immunoreactive to DSP antibody (Figs. 4G, 4H). Therefore, our inability to detect DSP expression on the matrix of newly formed reparative dentin-like tissue that clearly failed to show tubular dentin structure was not surprising (Figs. 4F, 4G). We used human-specific anti-mitochondrial immunohistochemistry to identify human cells and their patterns of distribution in the DPSC/dentin transplants. It was found that the dentinogenic cells either lining the surface of or trapped within newly formed reparative dentin-like structure were human in origin (Figs. 4I, 4J). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Development of stem-cell-mediated organ-like structure is a dynamic process that involves mutual interactions between transplanted stem cells and the host microenvironment. Eventually, the organ-like structure is a chimeric structure of donor and host cellular components. Here we show that bFGF, an important mitogen for vascularization and organogenesis (Bouma-ter Steege et al., 2001; Traver and Zon, 2002), is specifically up-regulated and may play an important role in not only ingrowth of blood vessels, but also the recruitment of hematopoietic elements in BMSSC transplants. MMP-9, an endothelial-cell-secreted matrix metalloproteinase (gelatinase B), is well-known for its ability to degrade collagen present in the vascular basement membrane (Murphy and Crabbe, 1995), and it plays a central role during angiogenesis (Nguyen et al., 2001). It is reasonable to speculate that up-regulated MMP-9 expression in BMSSC transplants contributes to angiogenesis and subsequent hematopoietic marrow formation. These processes are absent in DPSC transplants, although vascularization can be demonstrated by VEGF expression.
While DSP has been reported to be present not only in dentin but also in bone at very low levels (Qin et al., 2002), in our experiments it was highly expressed in the newly formed dentin but could not be detected by immunohistochemical staining in osteoblasts and bone matrix of BMSSC transplants (Gronthos et al., 2002). Although the mechanisms of how DSP contributes to dentinogenesis in DPSC transplants are not yet clear, it may be involved in the generation of a dentin/pulp complex in vivo.
Dentin does not remodel, but responds to an injury by forming reparative dentin to protect the dental pulp (Murray et al., 2000; About et al., 2001). It has been reported that bone sialoprotein and dentin matrix components are able to stimulate reparative dentin formation (Decup et al., 2000; Smith and Lesot, 2001) and that growth factors such as TGFßs and BMP-7 are capable of stimulating secretion of extracellular matrix by odontoblasts (Sloan and Smith, 1999; Sloan et al., 2000). However, the restoration of damaged dentin is still limited to conventional treatments such as fillings and crown restorations (Baum and Mooney, 2000; Kaigler and Mooney, 2001). Thus, regeneration of living teeth, or parts thereof, is considered to be a promising new therapeutic strategy. Unlike in the DPSC/HA/TCP transplants (Gronthos et al., 2000, 2002), DPSCs failed to generate an organized dentinal tubule structure in the DPSC/dentin transplants. This may be due to differences in the chemical composition and three-dimensional structure of HA/TCP and acid-treated dentin. Here we demonstrate that DPSCs are capable of forming reparative dentin-like structure on the surface of human dentin, indicating the possibility and challenge of using DPSCs to repair tooth structures and to seal root canals.
The mechanisms that control the distinct differentiation of BMSSCs and DPSCs in vivo are not fully understood at this time. This study provides the first evidence to demonstrate that bFGF/MMP-9 and DSP may play important roles in the formation of a bone/marrow organ structure and a dentin/pulp complex, respectively, suggesting that osteogenesis and dentinogenesis are controlled by different mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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authors contributing equally to this work; 
Received May 14, 2003; Last revision September 18, 2003; Accepted September 23, 2003
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