Isolation and Assessment of Mesenchymal Stem Cells Derived From Bone Marrow: Histologic and Histomorphometric Study in a Canine Periodontal Defect
The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 107 mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations.
(a) Marrow-derived cell culture. At primary culture, some cells tended to adhere on the culture surface. (b) The cells multiplied and the culture reached confluency at 10 days (Bar = 100 μm). (c) Flow cytometric analysis of the isolated cells from canine marrow. Majority of the cells tended to express mesenchymal markers including CD44 and CD90. Non-mesenchymal markers including CD146, SSEA-4, and anti-macrophage antigen were expressed in a small percentage of the cells.
Figure 2. Differentiation potential of isolated cells from canine marrow. (a) The osteogenic culture stained by alizarin red. (b) The adipogenic culture stained by Oil Red (Bar = 100 μm). Figure 3. Photomicrograph of a mesiodistal histologic section of one of the specimens in BMSC group including both reference notches. Thick new cementum (arrow) and new bone (asterisk) have been formed on the entire root surface between the notches. The PDL is well organized with collagen fibers oriented perpendicularly between the bone and cementum (double-chevron). Single chevron shows a blood vessel in the PDL (hematoxylin and eosin stain, original magnification ×40). Figure 4. Photomicrograph of a mesiodistal histologic section of one of the specimens in the control group, representing the apical notch. Arrow shows collagen fibers parallel to the root surface, and asterisk depicts new bone formation in the apical notch (hematoxylin and eosin stain, original magnification ×40; scale bar = 200 μm).
Bars show the percentage distribution of the values of the different parameters in test and control groups. PDL indicates periodontal ligament. *P < .05.
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