Osteogenic potential of Gingival Mesenchymal Stem Cells on Chitosan scaffold for Treatment of Peri-implant Defects (An Experimental Study)

.Introduction

Implant therapy is now considered an acceptable successful way to replace hopeless teeth ⁽¹⁾. Immediate implantation has many advantages like proper implant placement, decrease time needed for to receive prosthesis and patient satisfying with function and esthetics at time of implantation ^(2).

However, the main challenge of immediate post extraction implants is significant alveolar bone loss due to periodontal disease, abnormalities, traumatic injury, or physiological bone resorption. Many modalities are used nowadays to deal with these challenges. One of them the technology of tissue engineering that has proven to be a promising therapy for bone regeneration with the establishment of an artificial biomaterial containing regenerating-competent cells and osteogenic growth factors achievable ^(3).

Today, using of stem cells is considered as a mainstream strategy for periodontal therapy, particularly for complete regeneration of the periodontal complex, which implies not only the reconstruction of appropriate alveolar bone but also the induction of cementogenesis along the root surfaces with the oriented insertion of newly formed periodontal tissue ⁽⁴⁾.Regarding dental originated sources, gingival mesenchymal stem cells (GMSCs) considered more interesting alternatives to the other dental MSCs as they are much easier to get from the clinically resected gingival tissues. Therefore, it is of great concern to prove the multiple differentiation potentials of GMSCs for possible tissue engineering applications ⁽⁵⁾.

For tissue engineering, scaffolds are needed to provide a three-dimensional microenvironment to accommodate cells and guide their adhesion, growth, and subsequent differentiation. A plethora of materials have been developed for tissue engineering approaches, namely natural and synthetic polymers, metals, ceramics, and composites, which have been fabricated into porous scaffolds, microparticles, sponges, meshes, nanofibers, and gels.

Natural polymers are preferred over synthetic or metallic materials, since they have better biocompatibility and ability to degrade in vivo without releasing toxic substances ⁽⁶⁾. One of these natural polymers commonly used is chitosan which is biocompatible and biodegradable and is currently used with other polymers in a variety of tissue engineering applications.

Many studies revealed that using autogenous or xenogenous (human) mesenchymal stem cell MSC to treat peri-implant defects gives higher results in bone formation and bone implant contact than using synthetic bone graft alone.

Also, treatment of peri-implant defects with bone marrow derived MSC and bone marrow mononuclear cells that had undergone ex vivo osteogenic differentiation prior to clinical use ⁽⁷⁾ resulted in higher new bone apposition than scaffolds alone.

Materials and Method

A total of ten mongral dogs age ranging between 1 to 2 years old weight from 20 to 25kg were used in this study. All experiments were conducted in the animal house of the Faculty of Medicine, Cairo University, Egypt according to the recommendations and approval of the Ethics Committee on animal’s experimentation of the Faculty of Medicine, Cairo University.

• Isolation of gingival mesenchymal stem cell (GMSC):

Gingival sample will be obtained by resecting a small piece of gingiva to prepare gingival mesenchymal stem cell (GMSC). G-MSCs will be obtained from the healthy gingival collars around partially impacted third molars. Cells’ isolation and culture will be done as formerly described ^(8).

• Biodegradable scaffold synthesis: A biodegradable composite made of Chitosan (Ch) will be generated by a simple molding method as previously described ^(9).
• Animal preparation: The animal model will be prepared as previously described by Boix and colleagues ^(10).
• Surgical procedures: dogs will be anesthetized by intramuscular injection of ketamine (10 mg/kg) and xylazine (4 mg/kg), measures will be taken to minimize pain to the dogs. Under sterile conditions, the first mandibular premolar teeth will be extracted bilaterally. Immediately, distal bone defects adjacent to the mesial socket 6 mm in height, 4 mm in the bucco-lingual direction, and 5 mm in the mesio-distal direction were created in the mandible bone. Then titanium implants were installed into the mesial area of the bone defect in each side. The primary stability was assessed.
• Defects were randomly divided into 2 groups, control group in which defects were filled with chitosan scaffold alone and intervention group in which defects were filled with GMSCs carried on chitosan scaffold. Five dogs were sacrificed 1 month, and the others were sacrificed at 3 months post operation.
• Radiological evaluation was done by taking periapical radiographs at baseline, 1month and 3 months after implant placement according to time of scarification. Bone density was measured using Digora software.
• Data were collected, tabulated and statistically examined.

Fig.1 after extraction of 1^st          fig.2 after implant placement bilaterally

premolar bilaterally

Fig.3 placement of stem cell on   fig.4 placement of chitosan scaffold                              

 chitosan scaffold

Fig. suturing bilaterally

3.Results:

Paired sample t-test was done to compare bone density at baseline and after one and three months between control group and intervention group.

There was a significant difference in bone density change, as bone density change was higher in intervention group (mean= 58.73, SD= 11.00) than in control group (mean= 19.71, SD=2.60), p-value= 0.003.

No statistically significant difference was observed between the two groups regarding bone density change.

4. Discussion

Stem-cell-based therapies aiming at regeneration of bone defects have been a promising alternative for clinical trial ^(11). As novel postnatal stem cells, GMSCs have been paid extensive attention for their therapeutic potential in regenerative medicine ^(12).

GMSCs can be easily isolated from human gingival tissue which is usually discarded as biological waste in the clinic and proliferate rapidly in vitro to meet the transplantation requirement for cell amount.

The contribution of GMSCs to bone regeneration was confirmed by radiologic assessment of the bone density in the present study. As we found, at 1- and 3-months post transplantation, the newly formed bone in intervention group was significantly higher than that in control group at one month and it was also higher at 3 months at intervention than control but without significance.

In addition to the osteogenenic potential of GMSCs to promote the new bone forming, another possible explanation responsible for the therapeutic effects of GMSCs on bone injuries was the transplanted GMSCs triggered the endogenous MSCs recruitment which is known to be crucial for successful bone repair ^(13), though the mechanisms of MSCs recruitment to the injury sites were unclear.