Increased Bone Formation in a Porcine Critical Size Defect when using Hyaluronic Acid and TCP Coated Polycaprolactone Scaffolds Seeded with Autologous Dental Pulp Stem Cells

+1Jensen, J; 1Tvedesoe, C; 1Chen, M; 2Kraft, D C E; 3Nygaard, J V; 3Kristiansen, A A; 1Baas, J; 1Bünger C +1Orthopaedic Research Lab, Aarhus University Hospital, Aarhus, Denmark, 2Department of Orthodontics, School of Dentistry, Aarhus Univeristy, Aarhus,

Denmark, 3Interdisciplinary Nano Science Center (iNANO), Aarhus University, Aarhus, Denmark jj@ki.au.dk

INTRODUCTION: Recently, dental pulp tissue has been described as an alternative source for autologous adult mesenchymal stem cells (MSCs). As with bone marrow derived mesenchymal stem cells (BMSCs), these cells could be viable as a tool in bone tissue engineering, aiming to restore large bone defects. Dental pulp tissue is readily available as a result of the surgical removal of ectopically impacted third molars, and contains an accessible source of pulp-derived mesenchymal stem cells (DPSCs), which can be easily isolated and cultured. DPSC cultures have shown rapid growth with high proliferative rate in vitro. By seeding the stem cells pre implantation on a slow degrading, mechanically strong synthetic polymer, coated with a fast resorbable hydrophilic natural polymer and tricalcium phosphate (TCP), we hypothesized improved bone healing in a critical size defect. METHODS: The scaffolds were comprised of the polymer, polycaprolactone (PCL). The grid network of the scaffold was created by a rapid prototyping apparatus making a three dimensional grid structure by extruding the PCL with a final fiber diameter of 175 µm in a layer-by-layer deposition. The scaffolds were cylinder-shaped (15 mm diameter x 10 mm height) sized to press fit into the critical size defects. This scaffold is for future reference called Bioplotted. Afterwards, a new scaffold was created by infusing the bioplotted scaffold with 4 mg/ml hyaluronic acid + TCP (weight ratio: 10% hyaluronic acid) and afterwards freeze drying the scaffold. This procedure created a microporous structure within the bioplotted scaffold and coated the PCL to increase the hydrophilicity and adding the TCP to the surface of the scaffold (figure 1). This scaffold is for future reference called HT.

To investigate the in vivo potential of using autologous DPSCs on the polymer HT scaffold, a critical size porcine calvaria model was used. Furthermore, the osteogenic potential of DPSCs was compared to autologous BMSCs. The study was approved by the local Animal Care and Use Committee. A total of 13 skeletally mature 1-year-old Danish landrace pigs were used with termination 5 weeks post surgery. One month prior to surgery, bone marrow from the proximal femur and one molar tooth was extracted from each individual pig. Mononuclear cells were isolated from each extraction source and differentiated into osteogenic lineage using a Dulbecco’s Modified Eagle’s Medium (DMEM). A total of six non-penetrating holes were drilled in the calvaria. The size of the cylindrical defects were 10 mm in depths and 15 mm in diameter in accordance to the definition of critical size defect, and the defects were positioned at least 1 cm apart to avoid biological interaction.1 Three paired studies were chosen due to variability in the natural healing between the different levels in the calvaria observed in pilot studies (slowest natural healing in the anterior part of the calvaria). 5*10^6 cells from each cells source were seeded on HT scaffolds five days before surgery and used in one of the paired sub-studies. The scaffolds used in the three paired sub-studies is depicted on figure 2. After termination of animals, bone volume to total volume (BV/TV) was analyzed using µCT (Scanco Medical, Switzerland). A Cylindrical Region of Interest (ROI) of 15 mm in diameter and a depth of 8 mm, was chosen. BV/TV mean and significance between groups (t-test) was calculated using STATA 10 (StataCorp, TX, USA).

RESULTS SECTION: Random extra samples of the cell seeding on the scaffold was visualized by live/dead staining followed by confocal microscopy as well as SEM imaging (images not shown). The images displayed good cell adhesion and penetration of the scaffold. The µCT data showed significant more bone formation in the defect containing the HT scaffold compared to the empty defect (p=0,0468). HT scaffolds showed larger BV/TV when compared to the scaffold comprised of the bioplotted structure only (p=0,0002). When comparing the HT scaffolds seeded with autologous tem cells, the defect containing scaffolds seeded with DPSCs had a significantly higher BV/TV (0,0058). The BV/TV of the three paired studies are illustrated in figure 3.

DISCUSSION: DPSCs have shown lamellar bone forming capabilities similar to BMSCs in recent in vitro and small animal in vivo studies. This study show an osteogenic potential of the DPSCs superior to BMSCs which could be due to higher proliferation rate of DPSCs and dentin production. These findings could lead to a potential use of DPSCs in future bone tissue engineering in clinical settings. Preliminary in vitro optimization studies showed excellent DPSC proliferation and differentiation on the HT scaffolds. The Hyaluronic acid and TCP coating on the bioplotted scaffold results in higher BV/TV compared to the pure PCL scaffold, which could be due partly to the hydrophilic properties as well as cell migratory stimulus of the hyaluronic acid. With the rapid prototype manufacturing technique the clinician could acquire a custom made scaffold preseeded with autologous DPSCs within days. SIGNIFICANCE: This study helps bridging the gap between the experimental use of DPSCs and clinical application. By applying the cells to a biodegradable rapid prototyped scaffold and implanting them in a critical size large animal defect, we showed superior bone healing of the scaffold itself as well as when using DPSCs compared to BMSCs. REFERENCES 1 Schlegel et al. Biomaterials, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, 2006, PMID: 16831666

Figure 1. µCT 3D reconstruction and detailed SEM magnification of the Bioplotted scaffold (left) and the HT scaffold (right).

Figure 3. Box plot of BV/TV in the three studies. Top left: Empty scaffold compared to HT scaffold Top right: Bioplotted caompared to HT scaffolds Bottom left: BMSC+HT scaffold compared to DPSC+HT scaffold

Figure 2. Position and type of scaffolds in each of the three groups: Sub-study 1: HT scaffold and empty defect Sub-study 2: HT scaffold and bioplotted scaffold Sub-study 3: DPSCs (amount 5’10^6) seeded on HT scaffold and BMSCs (amount 5’10^6) seeded on HT scaffold

Poster No. 0614 • ORS 2012 Annual Meeting