Cell Therapy Based on Gingiva-Derived Mesenchymal Stem Cells Seeded in a Xenogeneic Collagen Matrix for Root Coverage of RT1 Gingival Lesions: An In Vivo Experimental Study

Abstract

(1) Background: To investigate the effect of a xenogeneic collagen matrix (CMX) seeded with autologous gingiva-derived mesenchymal cells (GMSCs) when combined with a coronally advanced flap (CAF) in the treatment of localized gingival recession type 1 (RT1). (2) Methods: Dehiscence-type defects were created in seven dogs. GMSCs were isolated, transfected with a vector carrying green fluorescent protein (GFP) and expanded. Once chronified, the defects were randomly treated with (1) CAF plus the combination of CMX and GFP⁺ GMSCs, (2) CAF plus CMX with autologous fibroblasts, (3) CAF plus CMX and (4) CAF alone. Histological and clinical outcomes at 2- and 6-week healing periods were analyzed and compared among groups. (3) Results: Histologically, the addition of autologous cells to the CMX resulted in reduced inflammation and a variable degree of new cementum/bone formation. CMX plus GMSCs resulted in greater mean recession reduction (1.42; SD = 1.88 mm) and percentage of teeth with recession reduction of ≥2 mm (57%) when compared to the other groups, although these differences were not statistically significant. (4) Conclusions: The histometric and clinical results indicated a positive trend favouring the combination of CMX and GMSCs with the CAF when compared to the groups without cells, although these differences were not statistically significant.

Keywords: cell therapy; gingiva-derived stem cells; gingival recession; mesenchymal stem cells; mesenchymal stromal cells; root coverage; soft tissue regeneration.

Figure 1

GMSC isolation, characterization and seeding into the matrix. GMSCs were isolated as described in Materials and Methods and expanded in tissue culture. (A) Phase-contrast micrography showing the exponentially growing GMSCs. These cells were then differentiated into fat cells (B), osteocytes (C) and chondrocytes (D). (E) Protocol describing the seeding of the undifferentiated GMSCs on the matrix; 2 × 10⁷ cells were dropped onto a matrix, incubated at 37 °C for 30 min, allowed to seed and then the matrix was transferred onto another well and media was added to cover the matrix and cells. Under the conditions used, between 50–60% of the cells were attached to the matrix.

Figure 2

Cross-sections of the buccal dento-gingival area after 2 weeks of healing. Haematoxylin and eosin (HE) stain. (a–d) Photomicrographs of the supracrestal periodontal tissues in the four groups. (e,f) Mild acute inflammatory infiltrate in the connective tissue in the CAF group. (g) Image showing root resorption with presence of osteoclasts in Howship’s lacunae in the same specimen from the CAF group. (h,i) Inflammatory infiltrate in the connective tissue surrounding the matrix in the CAF+CMX group. Note the high density of leucocytes and blood channels. (j,k) Collagen matrix within the connective tissue with mild inflammatory infiltrate and few fibroblasts in the CAF+CMX+GMSCs group and in the CAF+CMX+FBs group (l,m). Please note the lower density of leucocytes in the GMSCs and FBs groups in comparison with the CMX alone group. CAF, coronally advanced flap; CAF+CMX, coronally advanced flap+collagen matrix; CAF+CMX+GMSCs, coronally advanced flap+collagen matrix+gingiva-derived mesenchymal stem cells; CAF+CMX+FB, coronally advanced flap+collagen matrix+fibroblasts; SD, standard deviation; D, dentin; CT, connective tissue; CMX, collagen matrix; JE, junctional epithelium; black arrows, fibroblasts; red asterisks, blood channels. Scale bar = 100 μm.

Figure 3

Histological landmarks and histology slides of a tooth allocated to the GMSCs group at six weeks. (a) Landmarks used for histometric analysis; haematoxylin and eosin (HE) stain. (b) Cross-section of the buccal dento-gingival region; HE stain. (c) High-magnification images of the gingival area containing the matrix. (d) Masson’s trichrome stain. (e) Movat’s pentachrome stain. (f) Picrosirius red stain. (g,h,i) Immunohistochemistry for green fluorescent protein (GFP): control (g) and GFP-labeled samples (h). (i) High magnification image of the GFP-labeled sample. Note the different color intensity among the control and the GFP-labeled groups, demonstrating the influence of GFP⁺ cells on the formation of new periodontal tissues. M, the most coronal marginal mucosa; aJE, the most apical extent of the junctional epithelium; cC, the most coronal extent of the cementum; cN, the most apical portion of the coronal notch at the level of the gingival margin; aN, the most apical portion of the apical notch; Bc, the most coronal level of the bone crest; E; area where the enamel was before decalcification; D, dentin; CMX, collagen matrix; JE, junctional epithelium; CT, connective; FBs, fibroblasts; red asterisks, blood vessels. Scale bar = 0.1 mm (a); 1000 μm (b–g); 50 μm (c); 100 μm (i).

Figure 4

Bucco-lingual histological sections depicting the periodontal tissues at the level of the apical notch at 6 weeks; haematoxylin and eosin (HE) stain. (a–d) Images of the apical notch area in specimens from the four groups. (e,f) High-magnification images showing the newly formed cementum, bone and periodontal ligament in a specimen from the CAF group. (g,h) Images from a sample from the CAF+CMX group in which bone growth has extensively occurred despite the limited dimension of new cementum. (i) Newly formed connective tissue adhesion and cementum in a specimen from the GMSCs group. (j) New cementum formation in a resorption lacuna. (k,l) High-magnification pictures showing the histological attachment level gain associated with the newly formed cementum and the immature bone coronal to the apical aspect of the apical notch. CAF, coronally advanced flap; CAF+CMX, coronally advanced flap + collagen matrix; CAF+CMX+GMSCs, coronally advanced flap + collagen matrix + gingiva-derived mesenchymal stem cells; CAF+CMX+FBs, coronally advanced flap + collagen matrix + fibroblasts; SD, standard deviation; nB, new bone; nC, new cementum; nPDL, new periodontal ligament; C, cementum; D, dentin; aN, apical aspect of the apical notch; asterisks, blood vessels; black arrows, osteoblasts; yellow arrows, osteocytes; red arrow, cementoblast. Scale Bar = 100 μm.

Figure 5

Surgical interventions: (a) dehiscence-type defect creation; (b) root coverage procedure. Defect creation: (a) Scalloped incision, preserving 3 mm of keratinized tissue, followed by two horizontal incisions (2 mm) and two vertical releasing incisions extending 5 mm in the alveolar mucosa. (b) Removal of the buccal bone until reaching a vertical distance from the cemento-enamel junction to the mid-buccal bone of 5 mm. (c) Bone removal until showing the mesio-distal dimension of the root. (d) Flap sutured in an apical position. Root Coverage Surgery: (e) Design of the coronally advanced flap (4 weeks after defect creation) by performing two horizontal beveled incisions (at the level of the buccal cemento-enamel junction) at the mesial and distal aspects of the tooth with the recession defect followed by two vertical, slightly divergent releasing incisions. (f) Coronal notch performed at the level of the gingival margin and apical notch at the level of the bone crest, after flap-raising with a split–full–split approach; de-epithelialized papillae. (g) Suture of the collagen matrix with simple stitches. (h) Flap sutured in a coronal position.