Periodontal tissue regeneration by transplantation of autologous adipose tissue-derived multi-lineage progenitor cells

Abstract

Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Figure 1

Characteristics of adipose tissue-derived multi-lineage progenitor cells (ADMPCs). (a) Mesenchymal stem cell (MSC) surface marker expression on ADMPCs. The expression of each surface marker is shown on the shaded histogram. Staining with an isotype control mAb is represented by a black line. (b) Colony formation of ADMPCs. Cells were seeded with the indicated density and cultured for 2 weeks. Cells were then fixed and stained with 0.1% crystal violet. (c) Multipotency of ADMPCs. Representative images are shown of Alizarin red staining of ADMPCs cultured in osteogenic differentiation media for 28 days, Oil red O staining of ADMPCs cultured in adipogenic differentiation media for 24 days, and Toluidine blue staining of ADMPCs cultured in chondrogenic differentiation media for 20 days.

Figure 2

Representative images of adipose tissue-derived multi-lineage progenitor cell (ADMPC) transplantation. (a) Pre-operative intra-oral findings. This case was a 51-year-old female. The test tooth was an upper left second molar with a 6-mm periodontal pocket in the distal area. (b, c) Intra-oral findings during periodontal surgery. The flap operation was performed in accordance with the modified Widman procedure. After reversing the gingival flap, a circumferential intra-bony defect filled with granulation tissue was revealed and the granulation tissue was removed. After washing the intra-bony defect with normal saline, ADMPCs mixed with fibrin gel were transplanted into the intra-bony defect. (d) Postoperative intra-oral findings. Four weeks after ADMPC transplantation, no abnormal findings were found at the surgical site.

Figure 3

Clinical assessment of probing pocket depth (PD) and clinical attachment level (CAL). Results of (a) PD reduction and (b) CAL gain are shown as box-and-whisker plots. The box, notch, and horizontal line denote the distance between the first and third quartile ranges (interquartile range; IQR), mean, and median, respectively. The upper whisker indicates a maximum value smaller than 1.5 × IQR above the third quartile (Q3). Similarly, the lower whisker indicates a minimum value greater than 1.5 × IQR below the first quartile (Q1). It also displays the outliers which were defined as further outside than Q1 − 1.5 × IQR or Q3 + 1.5 × IQR. PD reduction and CAL gain of the test sites were measured at 3 months, 6 months, 9 months (n = 12). Individual data is shown in Supplemental Table 4. *p < 0.0001, with the use of a one-sample t-test based on the closed testing procedure.

Figure 4

Outcome of adipose tissue-derived multi-lineage progenitor cell (ADMPC) transplantation by dental radiographs. Radiographic outcomes of ADMPC-transplanted individuals. Case no. 2 was a 51-year-old woman. Case no. 10 was a 58-year-old woman. Dotted lines indicate the remaining alveolar bone crest or the bottom of the bone defect. The radiographs clearly show that the bone defect was filled with newly generated alveolar bone at 9 months after transplantation.

Figure 5

Clinical assessment of new bone formation. Results of new bone formation are shown using the same box-and-whisker plot representation as in Fig. 2. The new bone formation rate was measured using X-ray images at 1 month, 3 months, 6 months, and 9 months (n = 12). *p < 0.0001, ^†p < 0.001, ^‡p < 0.025, with the use of a one-sample t-test based on the closed testing procedure.