Potential Benefits from 3D Printing and Dental Pulp Stem Cells in Cleft Palate Treatments: An In Vivo Model Study

The interest in bone tissue engineering and regeneration
therapies, have grown in parallel with the increase in bone fractures
due to accidents, musculoskeletal disorders and congenital
diseases such as cleft palates [1-3].


Introduction
The interest in bone tissue engineering and regeneration therapies, have grown in parallel with the increase in bone fractures due to accidents, musculoskeletal disorders and congenital diseases such as cleft palates [1][2][3]. Cleft lips and/or palates are the most common congenital malformations of the head and neck and occur in the setting of multiple genetic and environmental factors [4]. In this sense, the repair of the cleft palate intends to establish the division between the oral and nasal cavity, thereby improving feeding, speech, and eustachian tube dysfunction, all while minimizing the negative impact on maxillary growth [5]. For example, although bone healing mediated by iliac crest has been very promising, its efficacy has not always been achieved. Perhaps this is due to the amount of implanted tissue or other factors but whatever the reason is, literature reports that after the treatment palatal wound dehiscence, a residual fistula has an estimated occurrence ranging from 3.4 to 27% [6] and other percentages [7,8]. With the advent of scaffolds printed in three dimensions (3D) [9], we can perhaps overcome those deficiencies. Scaffolds made by a 3D printer, having a microenvironment in three dimensions, may favor the comfort of cells, their survival and proliferation. In this paper the potential of pig dental pulp stem cell in combination with 3D scaffolds for becoming a choice for cleft palate defects treatments is examined in an in vivo model.

Scaffolds Manufacturing
The Polycaprolactone (PCL) used in this project was directly provided as a 1.75 ± 0,005 mm filament for standard 3D printers (3D4makers.com, Mw: 84500 ± 1000, 100% pure, Haarlem, Netherlands). For the fabrication of the scaffolds, a Hephestos 2 (Prusa i3 -BQ, Madrid, Spain) with a heated bed was used. The extruder was a double drive gear, with a noozle of 400 μm.

Immunophenotype of Pig Dental Pulp Stem Cells
The pig dental pulp stem cells phenotype was analyzed by flow cytometry, utilizing the following antibodies, coupled to fluorophores CD90-FITC, CD105 VB421. Previously, harvested stem cells were seeded in 6 wells plates, when the cells reached 80% confluence were placed in 1.5 ml eppendorf tubes, centrifuged at 600g. The cellular pellet was washed with phosphate buffer saline (PBS) supplemented with 2% of fetal calf serum and centrifuged again at 600g for 5 minutes. The cells were then resuspended in a 400 μl volume of PBS, supplemented with 2% FCS. before 0.3 μl of the two coupled antibodies were added without dilutions and then incubated for 15 min at room temperature before being read using the CytoFLEX LX flow cytometer (BRVYNI), selecting the corresponding filters for the detection of each of the fluorochromes with 10,000 events being captured for each sample. Pig dental pulp cells, without coupled antibodies, were used as controls.

Scaffold Sterilization and Seed of the Pig Dental Pulp Stem Cells
The 3D scaffold (Figure 1) was sterilized with ethanol at 70%, and then left in a physiological solution with antibiotic and antifungal properties for 24 hours. After this time it was placed in UV radiation for an additional 24 hrs, before the pig's dental pulp stem cells were planted in layers. First a layer, with 300 thousand cells, was placed in the scaffold, then a layer of Beta Tricalcium Phosphate powder (β-TCP) (R.T.R granules, Septodont, France) was added, thus 5 layers of each component were applied. The construct (3D scaffold + pig DPSC + β-TCP) was incubated for 48 hrs, before the surgery. The pig was anesthetized and a defect of 10 mm was created in the left side of the maxilla (Figure 2), the construct was placed in the defect ( Figure 3) and sutured with vicryl three zeros. After 6 months the pig was euthanized and the maxilla was removed. An occlusal X-ray was taken before the maxilla was subsequently placed in 4% formalin for histological analysis.

Conclusion
Stem cells from dental pulp are easy to obtain while also having the capacity to proliferate quickly. The use of 3D scaffolds, with osteo-receptor properties, helps to induce the regeneration of good quality bone in both the short and medium term.