Tissue Engineering with Compact Bone-Derived Cell Spheroids Enables Bone Formation around Transplanted Tooth

Nahomi Matsumura; Xianqi Li; Eri Uchikawa-Kitaya; Ni Li; Hongwei Dong; Kai Chen; Michiko Yoshizawa; Hideaki Kagami

doi:10.1007/s13770-021-00423-3

. 2022 Feb 4;19(2):377–387. doi: 10.1007/s13770-021-00423-3

Tissue Engineering with Compact Bone-Derived Cell Spheroids Enables Bone Formation around Transplanted Tooth

Nahomi Matsumura ^1,², Xianqi Li ^1,^2,³, Eri Uchikawa-Kitaya ^1,², Ni Li ^1,⁴, Hongwei Dong ¹, Kai Chen ^1,⁵, Michiko Yoshizawa ^1,², Hideaki Kagami ^1,^3,^6,^✉

PMCID: PMC8971212 PMID: 35119647

Abstract

BACKGROUND:

Although tooth transplantation is a desirable treatment option for congenital defects of permanent teeth in children, transplantation to a narrow alveolar ridge is not feasible. In this study, we investigated the possibility of bone tissue engineering simultaneously with tooth transplantation to enhance the width of the alveolar bone.

METHODS:

Bone marrow mononuclear cells or cortical bone-derived mesenchymal stromal cell spheroids were seeded onto atelocollagen sponge and transplanted with freshly extracted molars from mice of the same strain. New bone formation around the tooth root was evaluated using micro-computed tomography and histological analysis. Tooth alone, or tooth with scaffold but without cells, was also transplanted and served as controls.

RESULTS:

Micro-computed tomography showed new bone formation in the furcation area in all four groups. Remarkable bone formation outside the root was also observed in the cortical bone-derived mesenchymal stromal cell group, but was scarce in the other three groups. Histological analysis revealed that the space between the new bone and the root was filled with collagen fibers in all four groups, indicating that the periodontal ligament was maintained.

CONCLUSION:

This study demonstrates the potential of simultaneous alveolar bone expansion employing bone tissue engineering approach using cortical bone-derived mesenchymal stromal cell spheroids for tooth transplantation. The use of an orthotopic transplantation model may further clarify the feasibility and functional recovery of the transplanted tooth over a longer period.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13770-021-00423-3.

Keywords: Tooth transplantation, Cortical bone-derived mesenchymal stromal cells, Collagen, Scaffold, Bone tissue engineering

Introduction

Congenital absence of permanent teeth is occasionally observed, with a frequency of 0.15–16.2% [1]. The therapeutic options currently available include prosthetic treatment, prosthodontic treatment, and dental implant surgery [2–4]. Compared with other treatment options, tooth transplantation has several advantages. It does not require the preparation of adjacent teeth, which is mandatory for prosthetic treatment. Compared with prosthodontic treatment and dental implant surgery, tooth transplantation requires a shorter treatment period and is less costly [5]. Previous studies have shown a relatively good outcome; for example, the survival rate of transplanted tooth after 10 years was approximately 96.3% and complications, such as estimated ankyloses, root resorption, and pulp necrosis rates per year were 2.0, 2.9, and 3.3%, respectively [6]. In a systematic review, the prognosis of transplanted teeth after 6 years or more was reported to be 81% [7]. The survival rate of transplanted tooth after 10 years was reported to be 74% [8].

However, tooth transplantation has some disadvantages. The maxillary lateral incisors are among the most frequent sites of congenital tooth loss. Comparing the size of the alveolar bone with that of candidate donor tooth, such as premolars or molars, the width of the alveolar bone could be less and the ideal bone margin (> 0.5 mm) is not always available [9]. The lack of sufficient bone volume hampers the widespread use of tooth transplantation [10]. Accordingly, a treatment strategy to increase the bone width at the transplantation site is desirable.

For enabling transplantation to narrow alveolar ridge, bone augmentation, such as guided bone regeneration or bone transplantation in advance, at the transplantation site is conceivable. However, multiple operations are required, which impose a heavy burden on the patient. If bone augmentation can be performed simultaneously with tooth transplantation, the number of operations can be reduced, the treatment period can be shortened, and the burden on patients can be reduced. Currently, autologous bone and artificial bone transplantation are used for alveolar bone regeneration and ridge augmentation [11, 12]. Although the certainty of bone regeneration in autologous bone grafting is high, damage to healthy tissues is inevitable [13]. In contrast, in the cases of bone augmentation with bio-artificial bone substitutes, there are restrictions with regard to the shape of the defect site and the amount of regenerated bone, and the procedure is potentially associated with complications, such as wound rupture [11, 12]. Tissue engineering is a novel concept aimed at regenerating tissues using cultured cells, biodegradable scaffolds, and biological factors [14]. Bone tissue engineering for implant treatment has already been applied clinically [15, 16]. In a recent study from our group, we demonstrated the possibility of alveolar bone tissue engineering using a beta-tricalcium phosphate (β-TCP) block scaffold with bone marrow mononuclear cells (MNCs) simultaneously with tooth transplantation [17]. Besides scaffold materials, cell-laden hydrogels fabricated via 3D bioprinting have shown a great potential in alveolar bone tissue engineering [18, 19]. With the use of β-TCP block and MNCs, bone regeneration around the tooth was promoted; however, the effect of the MNC was obscure. In addition, the preparation of the β-TCP block to adapt to the complex tooth shape and bone defect was technically difficult. The results from this study suggested the necessity for more potent cells with higher bone regeneration capability and the use of scaffolds with excellent formativeness, such as hydrogels.

In this study, we employed spontaneous spheroids of cortical bone-derived mesenchymal stromal cells (CB-MSCs), which are reported to have excellent stem cell properties and high bone regeneration capability [20, 21]. As a scaffold, we selected an atelocollagen sponge, which possesses excellent formativeness and is readily used in dental clinics. The potential of simultaneous bone tissue engineering with the transplantation of developing tooth was tested using CB-MSC spheroids and atelocollagen sponge in an ectopic animal model.

Materials and methods

Animals

The experiments using animals in this study were performed in accordance with “The care and the use of laboratory animals for experimental procedures of the Matsumoto Dental University” guidelines with the approval of the “Matsumoto Dental University Committee on Intramural Animal Use” (approval number 269 and 318).

C57BL/6 J JmsSlc (C57BL/6 J) and C.B-17/IcrHsd-Prkdc^SCID (SCID) mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained in the laboratory animal facility of the Matsumoto Dental University High-Tech Center, Japan. Animals were kept in a temperature- and humidity-controlled environment with a 12 h light/dark cycle and provided free access to food and water.

Preparation of the atelocollagen scaffold

Atelocollagen sponge (8 mm in diameter; Olympus Terumo Biomaterials Co., Ltd., Tokyo, Japan) was cut into a semicircular shape, 5 mm in height. A cavity was formed using a needle to fit the shape of the tooth. The sponge was sterilized overnight by UV irradiation before use (Fig. 1). Before experiment, the biocompatibility of atelocollagen sponge was confirmed using 3T3 cells (Supplemental Fig. S1).

Fig. 1 — A schematic diagram of the experimental flow The maxillary first molar (M1) and second molar (M2) were extracted from 3-week-old male C57BL / 6 J mice. Subsequently, the tibia and femur were excised from the same animals, and mononuclear cells (MNCs) were extracted from the bone marrow. Cortical bone-derived mesenchymal stromal cells (CB-MSCs) were obtained from the remaining bone tips after enzymatic digestion and were cultured. Four groups were compared. In the tooth group, only the tooth was transplanted, without a scaffold. In the collagen group, the extracted tooth was embedded into the atelocollagen sponge and transplanted. In the MNC group, MNCs were seeded onto an atelocollagen sponge and transplanted. In the CB-MSC group, CB-MSC spheroids were seeded onto the atelocollagen sponge and transplanted. After 4 weeks, the transplants were excised and analyzed

Tooth extraction and preparation of MNCs

Tooth extraction and preparation of MNCs were performed according to the protocol reported by Uchikawa et al. [17]. Briefly, 3-week-old male C57BL/6 J mice were euthanized with an overdose of pentobarbital, administered through an intraperitoneal injection, and the maxillary first and second molars (M1 and M2) were extracted, at R3/4 stage of tooth formation [22]. The removal of blood and bone fragments was confirmed under a binocular stereomicroscope (Leica MZ6; Leica Mikrosysteme, Wetzlar, Germany) (Fig. 1).

The tibiae and femora were dissected, and their both ends were cut. After the bone marrow was flushed out with a syringe, MNCs were isolated using one-step density gradient centrifugation (Lymphocyte-Mammal, Cedarlane, Burlington, NC, USA) according to the manufacturer’s instructions, with modifications (centrifugation was performed at 800 × g instead of 400 × g for 30–40 min at 20 °C) [23].

Isolation and culture of CB-MSCs and spontaneous spheroid formation

CB-MSCs were isolated and cultured according to previously published protocols [24]. Briefly, the femurs and tibiae were used after the extraction of MNCs. Bone tissues were cut into 1–2 mm long fragments with scissors and incubated with 0.25% collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 45 min at 37 °C. Cells were washed with phosphate-buffered saline (PBS) and cultured in α-minimum essential medium (α-MEM, FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS, 1% penicillin–streptomycin-amphotericin solution, and 10 ng/mL recombinant human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ, USA). Passage 2 cells were collected and seeded into a 55 mm spheroid-forming low-adhesive culture dish (AS ONE, Osaka, Japan) at a density of 1.5 × 10⁴ cell/cm² according to our previous reports [18, 19]. CB-MSC spheroids formed after 24 h were stored in a cryoprotectant (5% DMSO + 95% FBS) at − 80 °C until use [21]. CB-MSCs cultured using this protocol were positive for mesenchymal stem cell markers, including CD29, CD105, CD51, and Sca-1 and negative for CD45 and CD11b (Supplemental Fig. 2) [21, 24]. When induced into the osteogenic cell lineage, CB-MSCs showed elevated ALP activity and were positive for ALP staining [24]. CB-MSCs showed fibroblastic cell morphology when cultured two dimensionally and the average size of spontaneous spheroids from CB-MSCs was 80.14 µm on an average [20]. When CB-MSCs spheroid-derived cells were cultured in an osteogenic induction medium for 1 week, the cells differentiated into osteogenic cells and expressed osteogenic markers, such as ALP, osterix, BSP, and DMP1, the levels of which were higher than those in monolayer-cultured BM-MSCs [20]. Although both monolayer-cultured CB-MSCs and CB-MSC spheroids possess bone forming capability in vivo [21], CB-MSC spheroids were used in this study considering their higher osteogenic differentiation capability [20].

Cell seeding and transplantation

A tooth was placed on the scaffold. Thereafter, MNCs (1 × 10⁶) or CB-MSC spheroids from the same number of cells in 50 µL of the culture medium were seeded directly onto the scaffold (MNC and CB-MSC groups, respectively). The same amount of culture medium was placed onto the scaffold without a tooth (collagen group). Tooth alone without a scaffold was also transplanted (tooth group).

Six-week-old male SCID (n = 28) mice were used as hosts for heterotopic transplantation. The samples were transplanted into four different areas on the back of SCID mice, as described previously [17]. The location for each sample group was randomly assigned. The mice were sacrificed 4 weeks after transplantation, and the transplants were harvested.

Micro-computed tomography (µCT) and morphometric analyses

The transplants were fixed in 4% buffered paraformaldehyde solution and scanned using micro-computed tomography (µCT: ScanXmate-A080; ComScan Tecno Co. Ltd, Yokohama, Japan). The methods for morphometric analyses were reported previously [17]. Briefly, the scanning parameters were as follows: voltage, 30 kV; current, 250 mA; and pixel size, 512 × 480. The area of the bone-like tissue was manually selected from each slice by a single examiner, according to the morphology. Once these CT images were reconstructed into three-dimensional (3D) images, morphometric analysis of the bone-like tissue was performed using an analytical software (TRI/3D-BON; Ratoc System Engineering Co. Ltd, Tokyo, Japan). The morphometric parameters used in this study included tissue volume (TV), bone volume (BV), bone volume fraction (BV/TV), bone surface (BS), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular spacing (Tb.Spac), trabecular bone pattern factor (TBPf), structure model index (SMI), and fractal dimension. TV outside the roots (TVo), BV/TV outside the roots (BVo/TVo), TV at the area between roots (TVr), and BV/TV in the area between roots (BVr/TVr) were also measured.

Histological and immunohistochemical analyses

After fixation and µCT imaging, the samples were decalcified with 10% neutral buffered ethylenediaminetetraacetic acid (EDTA, pH 7.1–7.5) for 14 days and embedded in paraffin. Samples were cut into 6 µm slices and stained with hematoxylin–eosin (HE) and Masson’s trichrome according to standard protocols.

For immunohistochemistry, anti-Sp7/osterix antibody (ab22552, Abcam, Japan) was used as the primary antibody and sections were incubated with it overnight at 4 °C. As a negative control, sections were incubated with 5% bovine serum albumin in PBS. The sections were then washed with PBS three times. ImmPRESS® Anti-Rabbit Ig (MP-7401, Vector Laboratories, Inc. Burlingame, USA) and ImmPACT DAB (SK-4105, Vector Laboratories, Inc. Burlingame, USA) were used for visualization according to the manufacturer’s instructions. The sections were counterstained with hematoxylin. Photomicrographs were captured using a Zeiss microscope (Axioskop 2 plus, Gottingen, Germany).

Statistical analysis

The Easy R (EZR) v3.4.1 software was used for statistical analysis [23]. Data are expressed as the mean ± standard deviation (SD). One-way ANOVA was used, and the Tukey–Kramer test was performed for multiple comparisons as a post-test. A value of p < 0.05 was considered statistically significant.

Results

Morphometric analyses using µCT images

The 3D reconstructed µCT images showed bone-like opaque tissues between the transplanted tooth roots in all groups (Fig. 2A-D). In the tooth, collagen, and MNC groups, the bone-like tissue was almost limited to an area between the roots (Fig. 2A-C), whereas in the CB-MSC group, it was also observed outside the roots and occasionally around the crown (Fig. 2D). TV, BV, and BS were not significantly different among the tooth, collagen, and MNC groups (Fig. 2E–G). In contrast, the values of these parameters were significantly larger in the CB-MSC group than in the other three groups (p < 0.01) (Fig. 2E-G). BV/TV was not significantly different among the groups (Fig. 2H). TVo was significantly larger in the CB-MSC group than in the other three groups (Fig. 2I). BVo/TVo was not significantly different among any of the groups (Fig. 2J). Similarly, TVr and BVr/TVr were not significantly different among any of the groups (Fig. 2K, L). When comparing the percentages of TVo and TVr, TVo was the largest in the CB-MSC group, showing a tendency to form bone-like tissue outside the roots (Fig. 2M).

Next, the trabecular structures were compared using µCT slice images. CT slice images showed a typical trabecular structure between roots in all groups, although only the CB-MSC group showed remarkable trabecular tissue outside the roots (Fig. 3A–D). The value of Tb.N in the tooth and collagen groups was significantly higher than that in the CB-MSC group (p < 0.05) (Fig. 3E). There was no significant difference in Tb.Th among the groups (Fig. 3F). The values of Tb.Sp and Tb.Spac in the tooth and collagen groups were significantly smaller than those in the CB-MSC group (p < 0.05) (Fig. 3G, H). Fractal dimension, SMI, and TbPf showed no significant differences among the groups (Fig. 3I–K).

Fig. 3 — Analyses of the trabecular structure using micro-computed tomography (µCT) slice and 3D images **A–D** µCT slice images of the four groups. E Comparison of trabecular number (Tb.N) among the four groups. Tb.N in the tooth and collagen groups was significantly higher than that in the CB-MSC group. F Comparison of Tb.Th among the four groups. G Comparison of trabecular separation (Tb.Sp) among the four groups. Tb.Sp in the tooth and collagen groups was significantly lower than that in the CB-MSC group. H Comparison of trabecular spacing (Tb.Spac) among the four groups. Tb.Spac in the tooth and collagen groups was significantly smaller than that in the CB-MSC group. I Comparison of fractal dimensions among the four groups. J Comparison of structure model index (SMI) among the four groups. K Comparison of trabecular bone pattern factor (TBPf) among the four groups. Coll: Collagen group. * p < 0.05

Histological and immunohistochemical analyses

HE staining revealed the formation of new bone (NB) mainly in the root furcation area in all groups (Fig. 4A-D); this was confirmed when the photomicrographs were observed at larger magnification (Fig. 4A-D). Interestingly, formation of NB, distant from the tooth, was observed only in the CB-MSC group (Fig. 4D, yellow arrow), and the newly formed bones and roots were not connected and divided by connective tissue (Fig. 4A-D). Outside the roots, connective tissue was also found around the root surface (Fig. 4A-D). In the CB-MSC group, newly formed bones were also observed outside the roots, and were divided by connective tissue (Fig. 4D).

Sp7-positive cells were distributed mainly in the vicinity of the newly formed bone (Fig. 4E-H). Interestingly, Sp7-positive cells were also distributed outside the roots, even in samples without the formation of NB (Fig. 4E-G), although the number of positive cells was less than that in the vicinity of newly formed bone (Fig. 4E-H).

To confirm the preservation of the periodontal ligament, the presence of collagen fibers was investigated using Masson's trichrome staining. In all four groups, strongly stained collagen fibers were observed at the furcation area, which arose from the cementum (Fig. 5A-D). Although most of the fibers were randomly aligned, there were some obliquely or vertically aligned fibers, connecting the newly formed bone and cementum-like native periodontal ligament. There were no differences in these tendencies among the four groups. Collagen fibers were also found outside the root, which arose from the cementum of the root surface (Fig. 5A-D).

Fig. 5 — Results of Masson's trichrome staining **A-D** Photomicrographs of Masson’s trichrome staining of the furcation area in the four groups. Most of the collagen fibers were randomly aligned, but there were some obliquely or vertically aligned fibers connecting the newly formed bone and cementum. Photomicrographs of Masson’s trichrome staining outside the roots from the four groups. Collagen fibers were also found in this area, and the fibers arose from the cementum of the root surface NB New bone, De Dentin. Scale bars: 100 µm

Discussion

In this study, an atelocollagen sponge was used as a scaffold for bone tissue engineering, simultaneously with tooth transplantation. Atelocollagen is a collagen without telopeptide, which is one of the causative factors of immunological reactions, and is widely used for medical and cosmetic purposes owing to its low antigenicity [26]. The atelocollagen used in this study was a sponge-shaped material obtained by mixing fibrotic atelocollagen and heat-denatured collagen at a ratio of 9:1 and cross-linked by heat treatment [27]. It has been used as a hemostatic material, wound dressing material, and as a scaffold for tissue engineering [28]. In particular, its formativeness and biocompatibility allow easy manipulation to fit a complex bone defect shape generated at the time of tooth transplantation and this was the reason we chose this material. Although atelocollagen sponge has been widely used as a scaffold for tissue engineering, its application in bone tissue engineering is limited due to its short absorption period. Until now, hydroxyapatite and β-TCP have been widely used as scaffolds for bone tissue engineering. Compared with the non-absorbable hydroxyapatite, β-TCP and its composites have been frequently used as scaffolds for bone tissue engineering because of their bioabsorbable nature [29]. β-TCP is known to last until 72 weeks after transplantation [30]; in contrast, the absorption period of collagen is approximately 4 weeks, which is extremely short. Moreover, collagen itself may inhibit bone formation [29, 31]. Accordingly, one of the major challenges in this study was the application of atelocollagen sponge as a scaffold for bone tissue engineering.

We observed that the collagen scaffold itself did not affect the bone formation around the transplanted tooth, which was clarified upon comparison of collagen and tooth groups. In contrast, when CB-MSC-derived spheroids were transplanted with a collagen scaffold, the formation of NB was significantly increased. In recent studies, it was shown that bone-forming cells in CB-MSCs have higher bone differentiation and bone-forming ability than bone marrow stromal cells, which have been widely used for bone regeneration [32]. Furthermore, spheroids obtained by three-dimensional culture of CB-MSCs were used in this study, which have higher bone differentiation and bone formation abilities than two-dimensionally cultured CB-MSCs [20, 21]. When atelocollagen sponge, with an extremely short absorption period, is used as a carrier for bone regeneration, it is conceivable that the use of highly osteogenic cells, such as CB-MSC spheroids, would be mandatory. In support of this notion, no significant increase in the formation of NB was observed in the MNC group compared with that in the collagen group. Because MNCs are a mixture of various types of cells and the proportion of bone marrow stem cells (CD271 + /CD45-) in human bone marrow MSCs is reported to be approximately 0.06% [33], the number of osteogenic cells might not be sufficient to enhance bone formation in this model.

In this study, cell tracking was not performed, which is one of the limitations of this study. Periodontal ligament cells have bone-forming ability [34], and it is also known that dental pulp contains highly osteogenic stem cells [35]. Accordingly, it is necessary to consider multiple cell sources for the origin of osteoblasts in this animal model. Because bone regeneration was observed in the area between roots in all groups, it can be speculated that the periodontal ligament attached to the surface of the extracted tooth played an important role in the formation of NB in this region. Moreover, in some samples, cementum-like enlarged hard tissue was formed around the apex of roots. Because this phenomenon was observed in all groups, it is also possible that the hyperplastic cementum- or bone-like tissue around the root apex might originate from osteogenic cells migrating from the dental pulp. New bones formed outside the roots were clearly more common in the CB-MSC group than in the other groups, suggesting that the newly formed bones outside the roots in the CB-MSC group originated from the transplanted CB-MSC spheroids. To confirm this hypothesis, we also transplanted CB-MSC spheroids or MNCs with atelocollagen sponge but without a tooth (Supplemental Fig. 3). Only CB-MSC spheroids with collagen sponge showed the formation of NB, which further supports the role of CB-MSC spheroids in enhancing the formation of NB only in the CB-MSC group.

When the microstructure of newly formed bone was analyzed, the CB-MSC group showed lower Tb.N, and higher Tb.Sp and Tb.Spac. This suggests that the NB in the CB-MSC group consisted of relatively sparse trabeculae compared with those in other groups. Interestingly, the slice images from µCT showed that the trabeculae between the roots were almost identical in all groups, but the newly formed bone outside the roots in the CB-MSC group was sparser than that between the roots. Because the formation of NB outside the roots was mostly found in the CB-MSC group, the smaller Tb.N and larger Tb.Sp and Tb.Spac values in the CB-MSC group may reflect the characteristics of the newly formed bone generated by CB-MSC spheroids. To ensure the usefulness of bone tissue engineering with a combination of CB-MSC spheroids and atelocollagen sponge, it is important to observe the maturation of newly formed bone at longer observation periods. In this study, we could only perform relatively short-term (4 weeks) observations using an ectopic transplantation model, which is another limitation of this study.

Preservation of the periodontal ligament is one of the most important factors in determining the success of tooth transplantation [9]. In this study, H-E- and Masson’s trichrome-stained sections showed the presence of dense collagen fibers between the newly formed bone and the roots, suggesting that the periodontal ligament was maintained after transplantation. However, most of the collagen fibers run parallel to the root surface, and only a small part of the functional structures connect the tooth root and the newly formed bone. Mechanical stimulation is required for the functional maturation of the periodontal ligament [36], and the lack of functional load in this ectopic transplantation model may not be sufficient to generate functional and mature periodontal ligaments. A long-term observation using an orthotopic model is required to confirm the maturation of the periodontal ligament.

In our previous study, we reported that tooth transplantation with a β-TCP block can enhance the formation of NB around the transplanted tooth, although simultaneous bone tissue engineering with MNC was not successful [17]. In terms of usability, the flexible atelocollagen sponge has a great advantage over the rigid β-TCP block, because it does not require a tremendous effort to shape the scaffold to fit tooth and bone defects. In addition, in this study, we show successful bone tissue engineering using atelocollagen sponge and CB-MSC spheroids simultaneously with tooth transplantation, which was not feasible with the combination of MNC and β-TCP block.

The results of this study show the potential of bone tissue engineering simultaneously with tooth transplantation, which is desirable for tooth transplantation to the narrow alveolar ridge.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (Tif 66 kb)^{(66.1KB, tif)}

Supplementary file2 (Tif 170 kb)^{(170.6KB, tif)}

Supplementary file3 (Tif 150 kb)^{(150.6KB, tif)}

Supplementary file4 (Tif 1154 kb)^{(1.1MB, tif)}

Declarations

Conflict of interest

The authors state that there is no conflict of interest regarding this manuscript.

Ethical statement

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1.Rakhshan V. Congenitally missing teeth (hypodontia): a review of the literature concerning the etiology, prevalence, risk factors, patterns and treatment. Dent Res J (Isfahan) 2015;12:1–13. doi: 10.4103/1735-3327.150286. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Bauss O, Sadat-Khonsari R, Engelke W, Kahl-Nieke B. Results of transplanting developing third molars as part of orthodontic space management. Part 1: clinical and radiographic results. J Orofac Orthop. 2002;63:483–92. doi: 10.1007/s00056-002-0131-4. [DOI] [PubMed] [Google Scholar]
3.Nagori SA, Bhutia O, Roychoudhury A, Pandey RM. Immediate autotransplantation of third molars: an experience of 57 cases. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;118:400–407. doi: 10.1016/j.oooo.2014.05.011. [DOI] [PubMed] [Google Scholar]
4.Atala-Acevedo C, Abarca J, Martínez-Zapata MJ, Díaz J, Olate S, Zaror C. Success rate of autotransplantation of teeth with an open apex: systematic review and meta-analysis. J Oral Maxillofac Surg. 2017;75:35–50. doi: 10.1016/j.joms.2016.09.010. [DOI] [PubMed] [Google Scholar]
5.Andreasen JO. Atlas of replantation and transplantation of teeth. 1. Tokyo: Quintessence Publishing Co., Ltd.; 1993. [Google Scholar]
6.Rohof ECM, Kerdijk W, Jansma J, Livas C, Ren Y. Autotransplantation of teeth with incomplete root formation: a systematic review and meta-analysis. Clin Oral Investig. 2018;22:1613–1624. doi: 10.1007/s00784-018-2408-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Machado LA, do Nascimento RR, Ferreira DM, Mattos CT, Vilella OV. Long-term prognosis of tooth autotransplantation: a systematic review and meta-analysis. Int J Oral Maxillofac Surg. 2016;45:610. doi: 10.1016/j.ijom.2015.11.010. [DOI] [PubMed] [Google Scholar]
8.Yang S, Jung BY, Pang NS. Outcomes of autotransplanted teeth and prognostic factors: a 10-year retrospective study. Clin Oral Investig. 2019;23:87–98. doi: 10.1007/s00784-018-2412-3. [DOI] [PubMed] [Google Scholar]
9.Andreasen JO. Histometric study of healing of periodontal tissues in rats after surgical injury. I. Design of a standardized surgical procedure. Odontol Revy. 1976;27:115–30. [PubMed] [Google Scholar]
10.Mejàre I, Stenlund H, Zelezny-Holmlund C. Caries incidence and lesion progression from adolescence to young adulthood: a prospective 15-year cohort study in Sweden. Caries Res. 2004;38:130–141. doi: 10.1159/000075937. [DOI] [PubMed] [Google Scholar]
11.Kagami H, Agata H, Satake M, Narita Y. Considerations on designing scaffold for soft and hard tissue engineering. In: Khang G, editor. The handbook of intelligent scaffold for regenerative medicine. Singapore: Pan Stanford Publishing; 2011. pp. 509–536. [Google Scholar]
12.Titsinides S, Agrogiannis G, Karatzas T. Bone grafting materials in dentoalveolar reconstruction: a comprehensive review. Jpn Dent Sci Rev. 2019;55:26–32. doi: 10.1016/j.jdsr.2018.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Starch-Jensen T, Deluiz D, Deb S, Bruun NH, Tinoco EMB. Harvesting of autogenous bone graft from the ascending mandibular ramus compared with the chin region: a systematic review and meta-analysis focusing on complications and donor site morbidity. J Oral Maxillofac Res. 2020;11:e1. doi: 10.5037/jomr.2020.11301. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Langer R, Vacanti JP. Tissue engineering. Science. 1993;260:920–926. doi: 10.1126/science.8493529. [DOI] [PubMed] [Google Scholar]
15.Kagami H. The potential use of cell-based therapies in the treatment of oral diseases. Oral Dis. 2015;21:545–549. doi: 10.1111/odi.12320. [DOI] [PubMed] [Google Scholar]
16.Shamsoddin E, Houshmand B, Golabgiran M. Biomaterial selection for bone augmentation in implant dentistry: a systematic review. J Adv Pharm Technol Res. 2019;10:46–50. doi: 10.4103/japtr.JAPTR_327_18. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Uchikawa E, Yoshizawa M, Li X, Matsumura N, Li N, Chen K, et al. Tooth transplantation with a β-tricalcium phosphate scaffold accelerates bone formation and periodontal tissue regeneration. Oral Dis. 2021;27:1226–1237. doi: 10.1111/odi.13634. [DOI] [PubMed] [Google Scholar]
18.Ma Y, Ji Y, Huang G, Ling K, Zhang X, Xu F. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix. Biofabrication. 2015;7:044105. doi: 10.1088/1758-5090/7/4/044105. [DOI] [PubMed] [Google Scholar]
19.Ma Y, Ji Y, Zhong T, Wan W, Yang Q, Li A, et al. Bioprinting-based PDLSC-ECM screening for in vivo repair of alveolar bone defect using cell-laden, injectable and photocrosslinkable hydrogels. ACS Biomater Sci Eng. 2017;3:3534–3545. doi: 10.1021/acsbiomaterials.7b00601. [DOI] [PubMed] [Google Scholar]
20.Chen K, Li X, Li N, Dong H, Zhang Y, Yoshizawa M, et al. Spontaneously formed spheroids from mouse compact bone-derived cells retain highly potent stem cells with enhanced differentiation capability. Stem Cells Int. 2019;2019:8469012. doi: 10.1155/2019/8469012. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Dong H, Li X, Chen K, Li N, Kagami H. Cryopreserved spontaneous spheroids from compact bone-derived mesenchymal stromal cells for bone tissue engineering. Tissue Eng Part C Methods. 2021;27:253–263. doi: 10.1089/ten.tec.2021.0001. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Moorrees CF, Fanning EA, Hunt EE., Jr Age variation of formation stages for ten permanent teeth. J Dent Res. 1963;42:1490–1502. doi: 10.1177/00220345630420062701. [DOI] [PubMed] [Google Scholar]
23.Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, et al. Differential stem- and progenitor-cell trafficking by prostaglandin E2. Nature. 2013;495:365–369. doi: 10.1038/nature11929. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Zhang Y, Li X, Chihara T, Mizoguchi T, Hori A, Udagawa N, et al. Comparing immunocompetent and immunodeficient mice as animal models for bone tissue engineering. Oral Dis. 2015;21:583–592. doi: 10.1111/odi.12319. [DOI] [PubMed] [Google Scholar]
25.Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48:452–458. doi: 10.1038/bmt.2012.244. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Miyata T, Taira T, Noishiki Y. Collagen engineering for biomaterial use. Clin Mater. 1992;9:139–148. doi: 10.1016/0267-6605(92)90093-9. [DOI] [PubMed] [Google Scholar]
27.Itoh H, Aso Y, Furuse M, Noishiki Y, Miyata T. A honeycomb collagen carrier for cell culture as a tissue engineering scaffold. Artif Organs. 2001;25:213–217. doi: 10.1046/j.1525-1594.2001.025003213.x. [DOI] [PubMed] [Google Scholar]
28.Ichioka S, Ohura N, Sekiya N, Shibata M, Nakatsuka T. Regenerative surgery for sacral pressure ulcers using collagen matrix substitute dermis (artificial dermis) Ann Plast Surg. 2003;51:383–389. doi: 10.1097/01.SAP.0000067971.90978.8F. [DOI] [PubMed] [Google Scholar]
29.Wei S, Ma JX, Xu L, Gu XS, Ma XL. Biodegradable materials for bone defect repair. Mil Med Res. 2020;7:54. doi: 10.1186/s40779-020-00280-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Ogose A, Kondo N, Umezu H, Hotta T, Kawashima H, Tokunaga K, et al. Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones. Biomaterials. 2006;27:1542–1549. doi: 10.1016/j.biomaterials.2005.08.034. [DOI] [PubMed] [Google Scholar]
31.Lang A, Kirchner M, Stefanowski J, Durst M, Weber MC, Pfeiffenberger M, et al. Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application. Acta Biomater. 2019;86:171–184. doi: 10.1016/j.actbio.2018.12.043. [DOI] [PubMed] [Google Scholar]
32.Fernandez-Moure JS, Corradetti B, Chan P, Van Eps JL, Janecek T, Rameshwar P, et al. Enhanced osteogenic potential of mesenchymal stem cells from cortical bone: a comparative analysis. Stem Cell Res Ther. 2015;6:203. doi: 10.1186/s13287-015-0193-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Henrich D, Verboket R, Schaible A, Kontradowitz K, Oppermann E, Brune JC, et al. Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro. Biomed Res Int. 2015;2015:762407. doi: 10.1155/2015/762407. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod Craniofac Res. 2007;10:149–160. doi: 10.1111/j.1601-6343.2007.00399.x. [DOI] [PubMed] [Google Scholar]
35.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625–13630. doi: 10.1073/pnas.240309797. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Kaku M, Rosales Rocabado JM, Kitami M, Ida T, Akiba Y, Yamauchi M, et al. Mechanical loading stimulates expression of collagen cross-linking associated enzymes in periodontal ligament. J Cell Physiol. 2016;231:926–933. doi: 10.1002/jcp.25184. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (Tif 66 kb)^{(66.1KB, tif)}

Supplementary file2 (Tif 170 kb)^{(170.6KB, tif)}

Supplementary file3 (Tif 150 kb)^{(150.6KB, tif)}

Supplementary file4 (Tif 1154 kb)^{(1.1MB, tif)}

[CR1] 1.Rakhshan V. Congenitally missing teeth (hypodontia): a review of the literature concerning the etiology, prevalence, risk factors, patterns and treatment. Dent Res J (Isfahan) 2015;12:1–13. doi: 10.4103/1735-3327.150286. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Bauss O, Sadat-Khonsari R, Engelke W, Kahl-Nieke B. Results of transplanting developing third molars as part of orthodontic space management. Part 1: clinical and radiographic results. J Orofac Orthop. 2002;63:483–92. doi: 10.1007/s00056-002-0131-4. [DOI] [PubMed] [Google Scholar]

[CR3] 3.Nagori SA, Bhutia O, Roychoudhury A, Pandey RM. Immediate autotransplantation of third molars: an experience of 57 cases. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;118:400–407. doi: 10.1016/j.oooo.2014.05.011. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Atala-Acevedo C, Abarca J, Martínez-Zapata MJ, Díaz J, Olate S, Zaror C. Success rate of autotransplantation of teeth with an open apex: systematic review and meta-analysis. J Oral Maxillofac Surg. 2017;75:35–50. doi: 10.1016/j.joms.2016.09.010. [DOI] [PubMed] [Google Scholar]

[CR5] 5.Andreasen JO. Atlas of replantation and transplantation of teeth. 1. Tokyo: Quintessence Publishing Co., Ltd.; 1993. [Google Scholar]

[CR6] 6.Rohof ECM, Kerdijk W, Jansma J, Livas C, Ren Y. Autotransplantation of teeth with incomplete root formation: a systematic review and meta-analysis. Clin Oral Investig. 2018;22:1613–1624. doi: 10.1007/s00784-018-2408-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Machado LA, do Nascimento RR, Ferreira DM, Mattos CT, Vilella OV. Long-term prognosis of tooth autotransplantation: a systematic review and meta-analysis. Int J Oral Maxillofac Surg. 2016;45:610. doi: 10.1016/j.ijom.2015.11.010. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Yang S, Jung BY, Pang NS. Outcomes of autotransplanted teeth and prognostic factors: a 10-year retrospective study. Clin Oral Investig. 2019;23:87–98. doi: 10.1007/s00784-018-2412-3. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Andreasen JO. Histometric study of healing of periodontal tissues in rats after surgical injury. I. Design of a standardized surgical procedure. Odontol Revy. 1976;27:115–30. [PubMed] [Google Scholar]

[CR10] 10.Mejàre I, Stenlund H, Zelezny-Holmlund C. Caries incidence and lesion progression from adolescence to young adulthood: a prospective 15-year cohort study in Sweden. Caries Res. 2004;38:130–141. doi: 10.1159/000075937. [DOI] [PubMed] [Google Scholar]

[CR11] 11.Kagami H, Agata H, Satake M, Narita Y. Considerations on designing scaffold for soft and hard tissue engineering. In: Khang G, editor. The handbook of intelligent scaffold for regenerative medicine. Singapore: Pan Stanford Publishing; 2011. pp. 509–536. [Google Scholar]

[CR12] 12.Titsinides S, Agrogiannis G, Karatzas T. Bone grafting materials in dentoalveolar reconstruction: a comprehensive review. Jpn Dent Sci Rev. 2019;55:26–32. doi: 10.1016/j.jdsr.2018.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Starch-Jensen T, Deluiz D, Deb S, Bruun NH, Tinoco EMB. Harvesting of autogenous bone graft from the ascending mandibular ramus compared with the chin region: a systematic review and meta-analysis focusing on complications and donor site morbidity. J Oral Maxillofac Res. 2020;11:e1. doi: 10.5037/jomr.2020.11301. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Langer R, Vacanti JP. Tissue engineering. Science. 1993;260:920–926. doi: 10.1126/science.8493529. [DOI] [PubMed] [Google Scholar]

[CR15] 15.Kagami H. The potential use of cell-based therapies in the treatment of oral diseases. Oral Dis. 2015;21:545–549. doi: 10.1111/odi.12320. [DOI] [PubMed] [Google Scholar]

[CR16] 16.Shamsoddin E, Houshmand B, Golabgiran M. Biomaterial selection for bone augmentation in implant dentistry: a systematic review. J Adv Pharm Technol Res. 2019;10:46–50. doi: 10.4103/japtr.JAPTR_327_18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Uchikawa E, Yoshizawa M, Li X, Matsumura N, Li N, Chen K, et al. Tooth transplantation with a β-tricalcium phosphate scaffold accelerates bone formation and periodontal tissue regeneration. Oral Dis. 2021;27:1226–1237. doi: 10.1111/odi.13634. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Ma Y, Ji Y, Huang G, Ling K, Zhang X, Xu F. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix. Biofabrication. 2015;7:044105. doi: 10.1088/1758-5090/7/4/044105. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Ma Y, Ji Y, Zhong T, Wan W, Yang Q, Li A, et al. Bioprinting-based PDLSC-ECM screening for in vivo repair of alveolar bone defect using cell-laden, injectable and photocrosslinkable hydrogels. ACS Biomater Sci Eng. 2017;3:3534–3545. doi: 10.1021/acsbiomaterials.7b00601. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Chen K, Li X, Li N, Dong H, Zhang Y, Yoshizawa M, et al. Spontaneously formed spheroids from mouse compact bone-derived cells retain highly potent stem cells with enhanced differentiation capability. Stem Cells Int. 2019;2019:8469012. doi: 10.1155/2019/8469012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Dong H, Li X, Chen K, Li N, Kagami H. Cryopreserved spontaneous spheroids from compact bone-derived mesenchymal stromal cells for bone tissue engineering. Tissue Eng Part C Methods. 2021;27:253–263. doi: 10.1089/ten.tec.2021.0001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Moorrees CF, Fanning EA, Hunt EE., Jr Age variation of formation stages for ten permanent teeth. J Dent Res. 1963;42:1490–1502. doi: 10.1177/00220345630420062701. [DOI] [PubMed] [Google Scholar]

[CR23] 23.Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, et al. Differential stem- and progenitor-cell trafficking by prostaglandin E2. Nature. 2013;495:365–369. doi: 10.1038/nature11929. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Zhang Y, Li X, Chihara T, Mizoguchi T, Hori A, Udagawa N, et al. Comparing immunocompetent and immunodeficient mice as animal models for bone tissue engineering. Oral Dis. 2015;21:583–592. doi: 10.1111/odi.12319. [DOI] [PubMed] [Google Scholar]

[CR25] 25.Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48:452–458. doi: 10.1038/bmt.2012.244. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Miyata T, Taira T, Noishiki Y. Collagen engineering for biomaterial use. Clin Mater. 1992;9:139–148. doi: 10.1016/0267-6605(92)90093-9. [DOI] [PubMed] [Google Scholar]

[CR27] 27.Itoh H, Aso Y, Furuse M, Noishiki Y, Miyata T. A honeycomb collagen carrier for cell culture as a tissue engineering scaffold. Artif Organs. 2001;25:213–217. doi: 10.1046/j.1525-1594.2001.025003213.x. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Ichioka S, Ohura N, Sekiya N, Shibata M, Nakatsuka T. Regenerative surgery for sacral pressure ulcers using collagen matrix substitute dermis (artificial dermis) Ann Plast Surg. 2003;51:383–389. doi: 10.1097/01.SAP.0000067971.90978.8F. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Wei S, Ma JX, Xu L, Gu XS, Ma XL. Biodegradable materials for bone defect repair. Mil Med Res. 2020;7:54. doi: 10.1186/s40779-020-00280-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Ogose A, Kondo N, Umezu H, Hotta T, Kawashima H, Tokunaga K, et al. Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones. Biomaterials. 2006;27:1542–1549. doi: 10.1016/j.biomaterials.2005.08.034. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Lang A, Kirchner M, Stefanowski J, Durst M, Weber MC, Pfeiffenberger M, et al. Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application. Acta Biomater. 2019;86:171–184. doi: 10.1016/j.actbio.2018.12.043. [DOI] [PubMed] [Google Scholar]

[CR32] 32.Fernandez-Moure JS, Corradetti B, Chan P, Van Eps JL, Janecek T, Rameshwar P, et al. Enhanced osteogenic potential of mesenchymal stem cells from cortical bone: a comparative analysis. Stem Cell Res Ther. 2015;6:203. doi: 10.1186/s13287-015-0193-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Henrich D, Verboket R, Schaible A, Kontradowitz K, Oppermann E, Brune JC, et al. Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro. Biomed Res Int. 2015;2015:762407. doi: 10.1155/2015/762407. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Gay IC, Chen S, MacDougall M. Isolation and characterization of multipotent human periodontal ligament stem cells. Orthod Craniofac Res. 2007;10:149–160. doi: 10.1111/j.1601-6343.2007.00399.x. [DOI] [PubMed] [Google Scholar]

[CR35] 35.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625–13630. doi: 10.1073/pnas.240309797. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Kaku M, Rosales Rocabado JM, Kitami M, Ida T, Akiba Y, Yamauchi M, et al. Mechanical loading stimulates expression of collagen cross-linking associated enzymes in periodontal ligament. J Cell Physiol. 2016;231:926–933. doi: 10.1002/jcp.25184. [DOI] [PubMed] [Google Scholar]

PERMALINK

Tissue Engineering with Compact Bone-Derived Cell Spheroids Enables Bone Formation around Transplanted Tooth

Nahomi Matsumura

Xianqi Li

Eri Uchikawa-Kitaya

Ni Li

Hongwei Dong

Kai Chen

Michiko Yoshizawa

Hideaki Kagami

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Supplementary Information

Introduction

Materials and methods

Animals

Preparation of the atelocollagen scaffold

Fig. 1.

Tooth extraction and preparation of MNCs

Isolation and culture of CB-MSCs and spontaneous spheroid formation

Cell seeding and transplantation

Micro-computed tomography (µCT) and morphometric analyses

Histological and immunohistochemical analyses

Statistical analysis

Results

Morphometric analyses using µCT images

Fig. 2.

Fig. 3.

Histological and immunohistochemical analyses

Fig. 4.

Fig. 5.

Discussion

Supplementary Information

Declarations

Conflict of interest

Ethical statement

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases