Int J Stem Cells.  2019 Mar;12(1):95-106. 10.15283/ijsc18074.

The Effect of Using Bone Marrow Mesenchymal Stem Cells Versus Platelet Rich Plasma on the Healing of Induced Oral Ulcer in Albino Rats

• Rashed FM
• GabAllah O
• AbuAli SY
• Shredah MT

• KMID: 2447228
• DOI: http://doi.org/10.15283/ijsc18074

Abstract

BACKGROUND AND OBJECTIVES
Oral ulceration is one of the most common debilitating condition that affects the oral cavity. In this study, the effect of locally injected platelet rich plasma (PRP) and bone marrow-derived mesenchymal stem cells (BMSCs) on the healing of oral ulcer was investigated.
METHODS AND RESULTS
An ulcer was induced in buccal mucosa of rats by using 5mm biopsy punch followed by application of cotton swab soaked with formocresol for 60sec. The ulcer was left untreated in the control group, treated with intralesional injection of PRP, or isolated cultured BMSCs. Data were analyzed clinically, histologically and immunohistologically on day 3, 5, 7 and 10. BMSCs group showed smaller ulcer area throughout the whole experimental period than the other groups with complete resolution of the ulcer on day 10, unlike the control group. However, there was no significant difference with PRP, on day 5, 7 and 10, regarding clinical ulcer size. BMSCs group showed better histological results regarding the rate of epithelial cell migration, the number of inflammatory cells, thickness and organization of collagen fibres and the number of blood vessels, with complete re-epithelization on day 10. BMSCs group showed a greater number of anti-PCNA positive nuclei throughout the whole experimental period than the other groups except on day 5, PRP had higher mean numbers of anti-PCNA positive nuclei in both tissues.
CONCLUSIONS
Both PRP and BMSCs accelerate wound healing and enhance the quality of the healing tissue with the latter being slightly more effective and faster.

Keyword

Formocresol; Induced oral ulcer; Mesenchymal stem cells; Platelet rich plasma; Rats; Wound healing

MeSH Terms

Animals
Biopsy
Blood Platelets*
Blood Vessels
Bone Marrow*
Collagen
Epithelial Cells
Injections, Intralesional
Mesenchymal Stromal Cells*
Mouth
Mouth Mucosa
Oral Ulcer*
Platelet-Rich Plasma*
Rats*
Ulcer
Wound Healing
Collagen

Figure

• 

  Fig. 1 Morphological characteristics of BMSCs using Phase-contrast inverted microscope. (A) Passage 2, 80% confluent showing homogenously fibroblastic like cell monolayer (×100). (Black arrows) showing cells undergoing mitosis. (B) Higher magnification of cells (×400) passage 2 showing spindle shaped fibroblast like cells with processes forming a monolayer. (C & D) FACS analysis for BMSCs at passage 3 demonstrated that 98.76% and 99.35% of the cultured cells expressed the mesenchymal CD44 and CD90 markers respectively, while they were negative for CD34 hematopoietic marker. These results indicated that relatively purified MSCs were isolated.

• 

  Fig. 2 (A) Clinical view of buccal ulcer of control group, PRP group, stem cell group at day 3, 5, 7 and 10 after ulcer induction. (B) Column chart representing the mean of ulcer surface area in mm2 in the control, PRP and stem cell group in the different time intervals (PRP, Platelet rich plasma).

• 

  Fig. 3 Representative histological images of ulcer healing in the control group (A~D), the PRP group (E~H) and the BMSCs group (I~L). Control group: Minimal epithelial migration above the dense granulation tissue on days 3 (A), 5 (B) and 7 (C). Persistence of a small ulcer area in epithelium with moderately inflamed underlying connective tissue on day 10 (D). PRP group: Deep epithelial cells migration at the wound margin above the dense granulation tissue on days 3 (E), 5 (F) and 7 (G). Complete wound closure on day 10 (H), the wound area is covered by thin layer of stratified squamous epithelium with thin keratin layer and flat rete ridges and the underlying connective still showing signs of moderate inflammation. BMSCs group: Epithelial proliferation and migration at the wound margin with moderate density of granulation tissue filling the wound core and has inflammatory cells infiltration on days 3 (I), 5 (J) and 7 (K). Full wound closure with complete re-epithelization on day 10 (L), the epithelium at the wound area showing maturation with moderate thickness of keratin layer and the underlying connective still showing signs of moderate inflammation. Haematoxylin-eosin staining; magnification, 40×. Scale bar=250 μm (PRP, Platelet rich plasma).

• 

  Fig. 4 Representative histological images of ulcer healing in the control group (A~D), the PRP group (E~H) and the BMSCs group (I~L). Control group: Collagen fibres at the wound area are thin and distributed in haphazard manner surrounded by numerous inflammatory cells, small new blood vessels and extravasated red blood cells on days 3 (A) and 5 (B). Combination of fine and moderately coarse collagen fibres that are randomly distributed with moderate inflammatory cells infiltration in addition to numerous small blood vessels on days 7 (C) and day 10 (D). PRP group: The central granulation tissue consists of fine interlacing collagen fibres, numerous dilated blood vessels and extravasated red blood cells on days 3 (E) and 5 (F). The connective tissue exhibiting thick collagen fibres with variable number of fibroblasts and numerous blood vessels on days 7 (G) and 10 (H). BMSCs group: moderately arranged interlacing collagen fibres running somewhat parallel to the surface epithelium, neovascularization and discrete inflammatory infiltration on days 3 (I) and 5 (J). The connective tissue starting to regain its normal architecture with more organized collagen fibres, many fibroblasts, high vascularity and mild inflammatory cell infiltration on days 7 (K) and 10 (L). Trichrome staining; magnification, 100×. Scale bar=100 μm (PRP, Platelet rich plasma).

• 

  Fig. 5 Left image, representative images of immunolocalization at wound area in the control group (A~D), the PRP group (E~H) and the BMSCs group (I~L). In the control group, relatively smaller number of nuclei showed immunoreactivity, in all the days compared to the PRP and BMSCs group. The BMSCs group showed a slightly higher number of positive nuclei than PRP group at the basal and suprabasal cells of epithelium and in some keratinocytes in migrating epithelium at ulcer surface. Similarly, the connective tissue cells, as well as endothelial cells lining the blood vessels of the BMSCs group, expressed a greater number of nuclear positive cells compared to the PRP and the control group. Anti-PCNA antibodies; magnification, 400×. Scale bar=25 μm (PRP, Platelet rich plasma). Right image, Column chart representing the mean of anti-PCNA antibodies positive nuclei in epithelial tissue (epi) in the control, PRP and stem cell groups in the different time intervals. Column chart representing the mean of anti-PCNA antibodies positive nuclei in connective tissue (CT) in the control, PRP and stem cell group in the different time intervals.

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