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British Orthodontic Society, Chapman Prize Winner 2003

A novel in vitro culture model to investigate the reaction of the dentine–pulp complex to orthodontic force

School of Dentistry, Birmingham, UK

Address for correspondence: A. A. Dhopatkar, Orthodontic Unit, School of Dentistry, St Chad’s Queensway, Birmingham, B4 6NN, UK. Email: dhopatkar{at}aol.com

Received March 4, 2004; accepted September 15, 2004

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Conclusions Authors and contributors References

 
Objective: To develop a novel mandible slice organ culture model to investigate the effects of externally applied force on the dentine–pulp complex.

Design: In vitro organ culture.

Setting: School of Dentistry, Birmingham, UK.

Materials and methods: Transverse 2 mm thick sections were cut from the mandibles of five 28-day-old male Wistar rats. Serial sections were used for control and test pairs. Springs made from 0.016-inch and 0.019 x 0.025-inch stainless steel wires were used to apply a 50 g tensile or compressive force, respectively, to test specimens. Control and test specimens were cultured for 5 days in a humidified incubator with 5% CO₂ at 37°C and processed for routine histological investigation. Nine more rats were used to provide control and compression test pairs where the pulps were extirpated after 3 days culture and total RNA isolated for gene expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR).

Results: Histology showed the dental and supporting tissues maintained a healthy appearance in the control cultures after culture. Histomorphometric analysis revealed a 20–27% increase in pulp fibroblast density in test specimens compared with controls. Gene expression analyses revealed up-regulation in the test groups of PCNA, c-Myc, Collagen 1, TGF-ß1 and alkaline phosphatase, whilst expression of osteocalcin was reduced.

Conclusions: The results demonstrated that the present organ culture technique provides a valuable in vitro experimental model for studying the effects of externally applied forces. These forces stimulated a cellular response in the pulp chamber characterized by altered gene expression and proliferation of fibroblasts; the latter being unaffected by the nature of the force in terms of compression or tension.

Key words: Organ culture, orthodontic force, dentine-pulp complex

	Introduction

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Continuing advances in orthodontic technology have highlighted a need to better understand the responses of the dental tissues to orthodontic forces. In particular, the effects on the dentine pulp complex have not yet been significantly investigated. Some workers¹ have found a mild inflammatory type response in the pulp of teeth that have been subjected to orthodontic forces, but specific cellular responses have not been investigated.

Orthodontic forces cause tooth movement, which results from a complex series of biological tissue reactions in response to the applied force. The resultant effect on the alveolar bone and other supporting structures of the tooth socket has been vigorously investigated at both the cellular and molecular level.^2–4

Relatively few studies have looked at reactions within the dental pulp.^5–7 Those that have been documented mainly show a trend towards the predominance of vascular changes. For example, application of an intrusive force to monkey incisors has been reported to cause necrosis of the pulp tissues.⁸ The effects of intrusive forces on human teeth have also been examined histologically.^9,10 The study sample consisted of 35 premolar teeth scheduled for orthodontic extraction from children aged 10–13 years. Sectional fixed appliances applied intrusive forces (35–250 g) for up to 35 days. Control teeth were usually obtained from different individuals to the test ones. Test teeth were extracted either immediately after the intrusive force was stopped or up to 104 days after force removal. The main pulp changes observed included vacuolization (inter-and intra-cellular spaces) and circulatory disturbances, (including enlarged and engorged blood vessels), local haemorrhage and brown haemosiderin deposits from red blood cell degradation. Root and dentine resorption were seen, as was a reduction of pre-dentine width in experimental groups, which was suggestive of a general degenerative change in the pulps. Pulp changes were not directly related to force magnitude, although a tendency towards greater severity with increased force was seen. Factors other than the magnitude of the force appeared to play a role with the developmental stage of the root being the most prominent. Other workers have noted vascular changes consequent upon orthodontic force application.¹

Not all workers have found degenerative changes in the pulps of teeth subjected to orthodontic forces. Nixon et al.¹¹ found a force-dependent increase in pre-dentine width at a time corresponding to the peak of the tooth movement cycle in rats. Indeed, even in the study by Stenvik and Mjör, where the overall results suggested that degenerative changes predominated,⁹ there were a few experimental teeth that showed increased activity in the odontoblast layer. This was accompanied by an increase in number of cells in the layer of Weil and a broader than usual pre-dentine layer.

There appears to be considerable variation in the observed pulpal response to orthodontic forces. It is difficult to compare different studies in view of the considerable variations in methodology, sample sizes and types. The nature and magnitude of force, however, as well as state of development of the root appear to be important variables that influence the response. In particular, many of the effects described above may be attributed to effects of circulatory changes brought about in response to applied forces. The specific biological mechanisms responsible for these vascular changes are as yet unclear. Enhanced angiogenesis has been reported to be due to diffusible angiogenic growth factors released from human dental pulp explants of orthodontically moved teeth.^12–14 Dentine matrix also contains growth factors, which may have angiogenic effects when released.¹⁵

Technological advances have continued to produce new materials, such as rectangular nickel titanium wires, and current approaches to orthodontic therapy may apply greater forces to teeth at earlier stages in treatment than was formerly the case. The need to understand the cellular and molecular mechanisms that protect the integrity, or affect behavior, of the dentine–pulp complex after such potentially iatrogenic insults thus assumes greater importance. An improved understanding of the mechanisms by which the integrity of the pulp is protected during orthodontic tooth movement may help provide a biologically sound basis for the optimization of clinical treatment strategies by harnessing the cellular activity of the dental pulp.

A tooth slice organ culture model has been previously described¹⁶ and applied to cytotoxicity testing for biomaterials.¹⁷ The aim of the present study was to develop a novel mandible slice organ culture model to investigate the effects of externally applied force on the dentine–pulp complex.

	Materials and methods

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The mandibles from freshly killed 28-day-old male Wistar rats were dissected out and immediately placed in sterile transport medium (Dulbecco’s Modified Eagle’s Medium, DMEM, Sigma Chemical Co., UK) at 37°C. Transverse sections of approximately 2 mm thickness were cut manually from each mandible using a diamond edged rotary saw (Isomet, Bühler Ltd., Germany) cooled with transport medium and pre-sterilized by washing with 70% aqueous ethanol. Each section was washed several times with medium and placed in sterile transport medium. Serial sections were labeled appropriately in order to provide comparable control and test pairs. To provide meaningful sample sizes, two animals were used for the tension experiment and three for the compression group, on the basis that each animal provides between 6 and 8 good sections.

Method for applying tension
A ‘ round’ steel rose-head bur in a rotary slow speed drill was used to create a hole in the bone of each test specimen using transport medium as coolant. The hole was positioned in the widest portion of the mandible at the root analogue end of the section and was used to allow one arm of the tension spring to be attached to the specimen (Figure 1a). Springs were constructed from 0.016-inch Australian stainless steel wire and both the springs and the test apparatus were sterilized by autoclaving using a 135°C cycle prior to use. After several washes in transport medium, the crown analogue end of the specimen was positioned and fixed to one end of the apparatus by means of a custom made clamp. The other arm of the tension spring was attached to one of a number of holes on the opposite side of the test apparatus. Force application was adjusted by varying the hole position employed. Springs were calibrated against a strain gauge to deliver a force of 50 g when 2 cm separated the two arms during the study.

View larger version (22K): [in this window] [in a new window]   Figure 1 Showing (a) tension spring set-up and (b) compression spring set-up

Organ culture technique
The culture medium consisted of DMEM containing 0.584 g/l glutamine and 1% penicillin/streptomycin solution (containing 100 units/ml of penicillin and 100 µg/ml of streptomycin), 20% heat inactivated fetal calf serum and 0.15 mg/ml ascorbic acid.

Each specimen was placed in an appropriate sized petri dish, immersed completely in the culture medium and cultured in a humidified incubator at 37°C in an atmosphere of 5% CO₂ in air. Daily changes of medium were carried out during the culture period.

After culture, each specimen was fixed for 24 hours in 10% (w/v) neutral buffered formalin (Surgipath, UK) and subsequently demineralized in 10% (v/v) formic acid (Sigma, UK) for 5 days before being processed for routine histological examination and embedded in paraffin wax (Tissue Tek III). Serial sections of 5 µm thickness were cut, and stained in Mayer’s hematoxylin and eosin (H&E). Five slides from each specimen in the compression experiment groups were stained with hematoxylin only to highlight the nuclei for the purposes of automated image analysis.

Histomorphometric analysis
Cell numbers in the pulps of cultured rat mandible slices were counted on 5 µm thick H&E-stained sections. Randomized manual cell counts were carried out under a x40 objective lens using a 10 x 10 square ocular graticule. Only one central row of 10 squares from the graticule was utilized for the cell counts and the graticule was placed on each slide overlying the midline of the central portion of the pulp at positions corresponding to the top, middle and bottom of the specimen. The total area inside the ten squares counted was 0.009 mm² (0.33 x 0.03 mm). Only clearly defined, well-stained nuclei were counted and partial nuclei were included in the count only if they were on the left or top of the grid. Nuclei were only counted once if they fell within two squares in the counting area. Mean cell counts were expressed as density per unit area.

For the tension test groups, five H&E-stained sections per specimen were selected for analysis using random number tables and six cell counts were carried out on each section, according to the protocol specified above.

Manual cell counts were performed initially for the compression group to determine whether there was a difference in cell numbers between test and control groups prior to confirmatory quantification using digital image analysis software. For this purpose three counts per H&E-stained section were carried out on one random slide per specimen.

Image analysis
Five haematoxylin-only stained slides from each compression group specimen were taken from the centre of the respective specimen blocks. A digital monochrome image of each slide was captured using Image Pro Plus image analysis software (Media Cybernetics, USA) with a x20 objective. At this magnification, the image represented most of the core area of the pulp for each slide. For this reason, only one cell count per slide was considered necessary when using image analysis. The software was programmed to count only those ‘nuclei’ that registered above a certain threshold level of density (intensity of staining) and within preset size parameters.

Statistics
The mean cell counts for control and test specimens were examined with a one-way analysis of variance (ANOVA) to determine if there was a statistical difference between the test and control groups.

To examine reproducibility of the manual counting technique, 10 of the tension group and 5 of the compression slides were re-counted after a period of 3 weeks. No significant difference was found between the two sets of counts using one-way ANOVA.

The mean areas for the structures recorded as ‘nuclei’ with image analysis in the compression test and control groups were compared using ANOVA, and no statistically significant difference was found. This confirmed that the software parameters had been correctly set to record nuclei.

All statistical analyses were carried out using the Minitab software package (Minitab Inc., USA).

Gene expression studies
RNA isolation and cDNA synthesis. Thirty-five paired control and test mandible sections were cut from 9 freshly killed 28-day-old male Wistar rats and cultured for 3 days. Test specimens were each subjected to a 50 g force from a compression spring during the culture period. The pulp from all control and test sections were extirpated with a blunt excavator instrument at room temperature and pooled into two separate groups. It was necessary to pool the extirpated pulps in this manner as preliminary studies showed that it was impossible to extract workable quantities of RNA from smaller samples. Total RNA was extracted from these samples using the RNeasy Mini Kit (Qiagen, UK), as recommended by the manufacturer. Subsequently, the DNase digested total RNA was used for oligo-dT reverse transcription to generate single stranded cDNA using the Omniscript kit (Qiagen, UK). Both RNA and cDNA concentrations were determined using a BioPhotometer (Eppendorf, UK).

Semi-quantitative reverse transcriptase polymerase chain reaction (Sq-RT-PCR analysis). Sq-RT-PCR analysis was performed for the genes PCNA, c-Myc, TGF-ß1, collagen 1, alkaline phosphatase and osteocalcin. PCNA and c-Myc were examined as indicators of cell proliferation, TGF-ß1 and collagen 1 as indicators of cellular activity and alkaline phosphatase and osteocalcin as genes involved in mineralization. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. A 50 ng sample of single stranded cDNA was used to seed a 50 µl PCR. Samples were amplified for between 22 and 30 cycles in an Eppendorf Master-cycler thermal cycler (Eppendorf, UK). Table 1 lists the primer sequences and cycle conditions used. Following the designated number of cycles, 8 µl of the reaction were removed and the product separated and visualized on a 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. Scanned gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalized against GAPDH control values. Each gene analysis was repeated twice and the average density value calculated was used in the final analysis.

	Results

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View larger version (105K): [in this window] [in a new window]   Figure 2 (a) H&E section showing viable bone, periodontal ligament and pulp in a 5 day cultured mandibular slice (x25). (b) Region of ‘normal’ periodontal ligament (x40)

View larger version (102K): [in this window] [in a new window]   Figure 3 Showing a. compressed region of periodontal ligament and b. region where periodontal fibres are stretched/under tension (x40)

View larger version (124K): [in this window] [in a new window]   Figure 4 Showing comparison of pulp fibroblast density in control (a) and test (b) specimens (x25)

View larger version (156K): [in this window] [in a new window]   Figure 5 Mosaic type appearance of test group pulps (x40)

View larger version (48K): [in this window] [in a new window]   Figure 6 Summarizing cell density data. (A) Tension group with manual cell counts. (B) Compression group with manual cell counts. (C) Compression group with image analysis

View larger version (30K): [in this window] [in a new window]   Figure 7 (a) Densitometric analysis of gel images after importing into AIDA image analysis software (Fuji, UK). (b) Gene expression data from Sq-RT-PCR analysis showing representative gel images and differential analysis of band intensity between control and test specimens expressed as percentage change. (c) All density calculations were relative to normalized GAPDH control values

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Conclusions Authors and contributors References

Increasing awareness of animal welfare has led to alternatives to in vivo animal experimentation being sought. A robust in vitro experimental model for studying the effects of orthodontic forces could provide such an alternative and importantly, allow better control of experimental variables.

Histological evidence from the present study shows that our mandible organ culture technique is viable and can be used for studying the effects of orthodontic forces on dental tissues, since the viability of the dental and supporting tissues were well maintained during the 5 day period in culture (Figure 2). In addition, the appearance of the supporting tissues in the specimens subjected to force (Figure 3) was similar to that described in previous studies.¹⁹ In particular, the areas of compressed or stretched periodontal ligament, which are central to the pressure–tension theory of orthodontic force transduction, were clearly visible. Whilst the present study concentrated on pulpal effects, one specific advantage of this in vitro approach is the ability to study all relevant dental and supporting structures together, including bone, periodontal ligament and pulp, and to examine their inter-relationships. In addition, the model allows the tissues to be investigated in isolation from vascular effects. The in vitro organ culture model therefore shows considerable potential for use in future studies.

The histomorphometric analysis (Figure 6), which revealed an increase in cell numbers in the core of pulps of test specimens subjected to both tension and compression is the first evidence that forces applied externally may be transmitted to the dental pulp to produce a cellular effect. The increase in cell numbers was found in the core or central portion of the pulp, and by location and appearance, it appeared that the applied force resulted in proliferation of pulp fibroblasts. It appears that the response of these cells is similar, regardless of whether tensile or compressive forces are applied. Considering the mechanisms by which external forces are transmitted to the dentine–pulp complex, certain parallels can be drawn between bone and dentine and, in particular, between odontoblasts and osteocytes. The odontoblast process, like the osteocyte, is completely encased in mineralized tissue. It is therefore possible that deformation of dentine may be ‘sensed’ by the odontoblasts, acting as mechanosensors, leading to activation of signaling mechanisms that produce cellular responses within the dentine–pulp complex. Similar effects have previously been implicated in the regulation of bone turnover.²⁰

Gene expression analysis was performed to investigate further the transduction mechanisms involved in the observed pulpal responses. In particular, the expression levels of proliferating cell nuclear antigen (PCNA) and c-Myc were examined. PCNA is a nuclear protein that is involved in DNA replication by acting as a cofactor for DNA polymerase and reaches its highest concentration during the S (DNA replication) phase of the cell cycle. It is an established marker of cell proliferation. c-Myc is an oncogene and altered expression of this gene generally results in alterations in the expression of its product, the Myc protein. Myc has been associated with the promotion of cellular growth and proliferation, as well as desensitization to growth inhibitory stimuli.²¹ Expression levels of both genes were elevated in test samples subjected to compressive force compared with controls (Figure 7). This result supports the initial histomorphometric findings and suggests that forces applied to teeth are capable of altering cellular behaviour at the molecular level. As initial results indicated that the response was independent of force direction, only compressive force was used, since it was easier to study.

Histological examination revealed a mosaic-like alteration of pulp extracellular matrix in teeth exposed to force (Figure 5) and gene expression data showed elevation of mRNA encoding collagen 1 (Figure 7). It is, however, unclear whether this altered appearance resulted from quantitative or conformational changes in the fibrous components. Up-regulation of collagen I gene expression in response to mechanical stress has been reported in other connective tissues.²² In addition, it has been shown in vitro that the multifunctional cytokine TGF-ß1 can stimulate collagen synthesis by fibroblasts.²³ The increased expression of TGF-ß1 in the present study (Figure 7) could therefore be related to the matrix changes. Such a link between TGF-ß1 and collagen synthesis after mechanical stress has been previously reported in anterior cruciate ligament fibroblasts.²² TGF-ß1 has also been implicated in tertiary dentinogenesis in response to injury.²⁴ Although not seen within the time span of the current study, an increase in pre-dentine width has been reported in rat teeth after sustained force application.¹¹ It is possible that the increased TGF-ß1 expression seen in the present study may be associated with such effects. The up-regulation of the enzyme alkaline phosphatase (Figure 7), known to be important in mineralization events, may also be related. However, the concomitant finding that expression of the gene encoding the mineralized matrix component osteocalcin was down-regulated seems incongruous in this context and requires further investigation.

The gene expression studies were carried out on extirpated rat pulp tissue. Recent evidence suggests that, when rat pulp is extirpated at room temperature, the odontoblasts lining the pulp cavity are also removed with the pulp.²⁵ The heterogeneous nature of the cell population therefore makes it impossible to attribute effects to specific cell types. It may be, for example, that the up-regulation of TGF-ß1 could be related to odontoblast activity, rather than to the pulpal fibroblasts.

The present study provides a novel in vitro experimental model for study of tissue responses to applied orthodontic forces, but care is required in direct extrapolation of these data to the clinical situation where force levels may differ. It is unclear as to what proportion of the applied force was actually transmitted to the dentine–pulp complex, but this study has highlighted the potential cellular effects of forces applied to the tissues and further investigations on the details of force transmission with this model should prove valuable.

	Conclusions

Top Abstract Introduction Materials and methods Results Discussion Conclusions Authors and contributors References

 
A novel in vitro organ culture model has been developed to study the effects of orthodontic forces on dental tissues and its application to study of the effects of external forces on the dentine pulp complex has been described. From the initial studies using this model, the following specific conclusions may be drawn:

• Externally applied forces can be transmitted to the dentine pulp complex.
  
• Externally applied forces can produce a cellular response in the pulp chamber, which is characterized by proliferation of fibroblasts and is unaffected by the nature of the force in terms of compression or tension.
  
• Force application alters gene expression within the dentine–pulp complex. In particular, genes associated with cellular proliferation (e.g. PCNA) and the extra cellular matrix component collagen 1 are up-regulated.
  

	Authors and contributors

Top Abstract Introduction Materials and methods Results Discussion Conclusions Authors and contributors References

 
Professor A. J. Smith, Drs A. J. Sloan, P. R. Cooper and W. P. Rock all contributed substantially to conception and design, analysis and interpretation of data, drafting and critical revision of the paper, and on final approval of the published version. Dr A. A. Dhopatkar was also responsible for carrying out the laboratory work and data collection and is the guarantor.

We thank Mr E. Harrington for his expertise in construction of the tension test apparatus and Dr J. B. MATTHEWS for his help with the digital image analysis work.

	Acknowledgments

 
This work was supported by the British Orthodontic Society Foundation.

	References

Top Abstract Introduction Materials and methods Results Discussion Conclusions Authors and contributors References

 
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dhopatkar, A. A. Articles by Smith, A. J. Search for Related Content PubMed PubMed Citation Articles by Dhopatkar, A. A. Articles by Smith, A. J.