Application of stem cells in the orthodontic maxillary expansion

Abstract

This invention relates to the contributions of the bone marrow—and adipose tissue—derived mesenchymal stem cells applied following the maxillary expansion—a frequently used method for the orthodontic treatment—to the healing of the expanded bone tissues. In the invention, adipose tissue and bone marrow-derived stem cell applications increased the healing and quality of the bones following the maxillary expansion and the applied stem cells were involved in the bone structure. Thus, the relapse following the maxillary expansion treatment was avoided. This application will shorten the treatment period for patients undergoing the maxillary expansion treatment and the patients will not have to use apparatus for a long period of time.

Claims

1. A method for an orthodontic maxillary expansion treatment for a patient, consisting of: isolating a plurality of stem cells; expanding maxillary; and injecting the stem cells into a midpalatal suture region of the patient.

2. The method of claim 1, wherein the stem cells are bone marrow derived stem cells.

3. The method of claim 1, wherein the stem cells are adipose tissue derived stem cells.

4. The method of claim 1, wherein the method further includes injecting the stem cells into the midpalatal suture region at a dose of 1 million cells.

5. The method of claim 3, wherein the stem cells are adipose tissue derived mesenchymal stem cells (ATMSCs).

6. The method of claim 2, wherein the stem cells are bone marrow derived mesenchymal stein cells (BMMSCs).

7. The method of claim 3, wherein the method further includes injecting the stem cells into the midpalatal suture region at a dose of 1 million cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The figures showing the results obtained from the experimental studies conducted in order to reach the goal of this invention are enclosed and are as follows:

(2) FIG. 1—In vitro morphological appearances of ATMSCs (a) and BMMSCs (b).

(3) FIG. 2—Osteocalcin staining: a. ATMSCs, b. BMMSCs; Collagen type 1 staining, c. ATMSCs and d. BMMSCs—The appearances of the differentiation experiment results of ATMSCs and BMMSCs.

(4) FIG. 3—a. ATMSCs and b. BMMSCs—The appearances of premaxillary tissue under fluorescence microscopy.

(5) FIG. 4—A. Osteoblasts (ob), osteoclasts (oc) and vessels (v) formed through the application of BMMSCs in the mid-sutural region. B. Osteoblasts (ob), osteoclasts (oc) and vessels (v) formed through the application of ATMSCs in the mid-sutural region; the appearances of the new bone formations in the mid-palatal suture region.

(6) FIG. 5—The appearance of the histomorphometric analysis of the new bone formation in the mid-palatal suture region; a. The comparison of the bone formation in the suture region; b. The comparison of the number of osteoblasts in the suture region; c. The comparison of the number of vessels in the suture region.

DETAILED DESCRIPTION OF THE INVENTION

(7) Experimental Study

(8) The experimental study and its results providing a basis for the developed model are indicated below.

(9) The Isolation and Characterization of Stem Cells

(10) The isolation of BMMSCs and ATMSCs from 3-month-old Wistar rats was conducted with known applications in the technique (16, 17). In order to characterize the obtained stein cells, they were analyzed through flow cytometry and then, their in vitro bone-forming potentials were shown.

(11) Maxillary Expansion in Rats

(12) 30 male Wistar rats were distributed in three equal groups. In each group, one single helix of 2 mm in diameter bent from stainless steel wire of 0.014 inches and a spring with arms of 10 mm each were used to expand mid-palatal suture. The activation was made on a millimetric paper and was adjusted to apply a strength of 30 grams.

(13) Under general anesthesia, upper two incisors were drilled with holes on front and hack sides at the level of gingival edges (papilla) and the expansion apparatus was placed into these holes from the front of the teeth.

(14) Injection of the Stem Cells

(15) Twenty-four hours following the placement of the expansion apparatus, ATMSCs in the group 1, BMMSCs in the group 2 and normal saline in the group 3 (control group) were injected into the mid-palatal suture region. Stem Cells were prepared in 100 μl of PBS (Phosphate-Buffered Saline) and then injected using insulin needles into the mid-palatal suture region (1 million cells per rat). Following an expansion period of five days, the expansion apparatus was removed and brackets were placed in the holes drilled on the incisors for reinforcement. Following a reinforcement period of 10-14 days, the animals were sacrificed by applying high doses of anesthetics.

(16) Histomorphometric Assessment

(17) For the evaluation of hard tissue, the upper jaws of ten animals each from the control and experimental groups were dissected (elimination of the soft tissues) and fixated in a solution of 10% formalin. After the fixation, the brackets used for the reinforcement purposes were removed and the premaxilla was decalcified in 5% formic acid. The sections were obtained at vertical angles to the sagittal plane. The reference points were determined as the apex and 4 mm apical to the alveolar crest. The line passing through the reference planes is along the centre of the incisor crown at the level of gingiva. The samples were embedded in paraffin blocks and serial sections of 5 μm thickness were obtained. The histological sections were stained with hematoxylin-eosin and then the histomorphometric assessment was conducted.

(18) In Vivo Tracking of the Transplanted Stem Cells

(19) In order to determine the vitality of transplanted stem cells in the mid-palatal suture region, the bone marrow- and adipose tissue-derived stem cells were marked with PKH167 green (Sigma) cell membrane dye and then injected into the rats. After the conclusion of the experiment, the premaxilla dissected from the rats was frozen on dry ice and then serial sections were obtained with cryotome. The obtained sections were fixated and examined under fluorescence microscopy, and then the presence of the marked stem cells was identified.

(20) Results of the Experiment

(21) The stem cells obtained from 3-month-old Wistar rats were demonstrated to be similar to MSCs (Mesenchymal Stem Cells) through flow cytometry (Table 1) and differentiation experiments.

(22) TABLE-US-00001 TABLE 1 The characterization of ATMSCs and BMMSCs through flow cytometry

(23) The results of the flow cytometry showed that ATMSCs and BMMSCs are positive for MSC markers and negative for hematopoietic markers.

(24) When ATMSCs and BMMSCs were examined under in vitro microscope, it was observed that they had classic MSC morphology (FIG. 1).

(25) The potentials of ATMSCs and BMMSCs to differentiate into bone cells were shown and it was also shown that these cells produced bone markers—collagen type 1 and osteocalcin proteins—as a result of the differentiation (FIG. 2).

(26) 10-14 days following the application of ATMSCs and BMMSCs into the rats that had undergone maxillary expansion, the dissected premaxillary tissue was examined under the microscope and the presence of the marked stem cells was identified in the premaxillary tissue (white areas are injected cells a. ATMSCs and b. BMMSCs) (FIG. 3).

(27) The newly formed osteoblasts (ob), osteoclasts (oc) and vessels (v) in the mid-palatal suture region in the groups that had undergone stem cell application were identified through the histological assessment (FIG. 4). The histomorphometric assessment of the new bone formation in the mid-palatal suture region showed that the stem cell application provided a more effective bone formation compared to the negative control group (in which only normal saline was applied). The application of ATMSCs and BMMSCs enabled faster bone formation in the mid-palatal suture region following the maxillary expansion, and it also increased the number of osteoblasts and vessels in the mid-palatal suture region (FIG. 5).

(28) The fact that it was experimentally proved that the application of ATMSCs and BMMSCs accelerated the new bone formation in the mid-palatal suture region and that those stem cells were directly involved in the bone structures through the process of new bone formation paved the way for the new treatment methods. This invention with the obtained findings can be applied to all mammals including humans.

(29) The shortening of the orthodontic treatment period with stem cell therapies, reduction of the relapse rate and thus achieving more stable results and improving the comfort of both patients and physicians are the advantages of this invention. The orthodontic treatment period, which gives a hard time for the patients since having to wear an apparatus during the treatment is esthetically unpleasant and also causes discomfort in their mouths, will be shortened with this application.

(30) Furthermore, the discomfort the patients are having will be relieved and a healthy relationship of the teeth to the jaw will be established. In addition to these, the completion of the treatment will be ensured to take place at a high level of patient cooperation.

(31) Thanks to this invention, bone marrow- and adipose tissue-derived stem cells applied following the maxillary expansion—a frequently used method for the orthodontic treatment—made favorable contributions to the healing of the expanded bone tissue. The relapse occurring after the maxillary expansion treatment can be avoided by speeding up the bone healing time and increasing the quality of the bones. Thanks to this application, a shorter treatment period for patients undergoing the maxillary expansion treatment will be ensured and the patients will not have to use apparatus for a long period of time.