Are Stem Cells Useful in the Regeneration and Repair of Cartilage Defects in the TMJ Condyle? An In Vivo Study

Research Article

J Dent & Oral Disord. 2021; 7(2): 1159.

Are Stem Cells Useful in the Regeneration and Repair of Cartilage Defects in the TMJ Condyle? An In Vivo Study

Guastaldi FPS1*, Hakim MA2#, Liapaki A1, Lowe B1, Faquin WC3, Thamm JR1 and McCain JP1

1Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Harvard School of Dental Medicine, Boston, MA, USA

2Department of Oral and Maxillofacial Surgery, University of Michigan, Ann Arbor, MI, USA

3Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

#Contributed Equally

*Corresponding author: Fernando Pozzi Semeghini Guastaldi, Skeletal Biology Research Center, Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Harvard School of Dental Medicine, 50 Blossom St, Thier Research Building, 513A, Boston, MA 02114, USA

Received: March 30, 2021; Accepted: April 14, 2021; Published: April 21, 2021

Abstract

Temporomandibular Joint (TMJ) disorders affect up to 10-40% of the population and if left untreated, may eventually lead to Osteoarthritis (OA) of the TMJ. In vivo TMJ repair and regeneration has received significant attention and represents a promising approach for the treatment of degenerative TMJ disorders. The aim of this study was to present a pre-clinical mouse model of TMJ articular cartilage defect and evaluate the utility of a potential tissue engineered TMJ therapy utilizing Mesenchymal Stem Cells (MSCs) derived from mice condyle, hydrogel, and biosilica. C57BL/6 mice (n=30) were equally divided into the following groups: sham group (S-group); control group, condylar cartilage defect only (CD-group); experimental group, condylar cartilage defect + direct administration of MSCs+hydrogel+biosilica (H-group). Mice were euthanized at 4 (n=15) and 8 (n=15) weeks and TMJ joint specimens were harvested for analysis. H&E and Safranin O stained sections showed intact articular surfaces on the condyle and glenoid fossa at both time points, maturation and distribution of chondrocytes along the condyle for the H-group compared to the CD-group. Data from this preliminary study shows that MSCs+hydrogel+biosilica may represent an experimental therapeutic compound for TMJ condylar cartilage regeneration.

Keywords: Temporomandibular joint; Tissue engineering; Hydrogel; Biosilica; Stem cells

Introduction

The Temporomandibular Joint (TMJ) plays a pivotal role in the movement coordination of the jaw during daily basic functions (i.e. speech, swallowing, eating). TMJ is a bilateral synovial joint composed of muscles, ligaments, the fibrocartilaginous articular surfaces of the mandibular condyle and glenoid fossa as well as the cartilaginous articular disc [1]. It is estimated that Temporomandibular Joint Disorders (TMDs) affect up to 10-40 % of the population, mainly young adults under 45 years of age with a sex ratio of 4:1 (women:men) or more among clinical cases of TMD pain [2], and among them 10% suffer from Osteoarthritis (OA) in the TMJ [3].

Left untreated, TMDs may eventually lead to OA of the TMJ [3]. TMJOA is typically a slowly progressive, inflammatory disease resulting in the degeneration of articulating tissues of both cartilage and subchondral bone [4] and presents either as asymptomatic1 or characterized by pain, limited function, and crepitus or clicking sounds [1,4,5].

Within the normal and healthy joint, the articular cartilage is indispensable for the smooth movements of the mandible coupled with the temporal bone bilaterally, guaranteeing a painless motion [3,6]. Clinical studies have reported that injury to the disc might be the most prevalent causative factor of TMJOA [7].

Several studies had effectively produced a degeneration resembling the OA by creating a defect on either the articular disc or the articular surface of the condyle. A partial discectomy on a mouse model and a disc perforation on rabbits, resulted in articular cartilage degeneration in both cases and early-onset of OA [7,8]. The latter model demonstrated also a heterotopic ossification in some animals with injured disc after 2-3 months [8]. Partially and totally removal of the condylar fibrocartilage in mice have been shown in recent publications to induce ectopic bone tissue and osteophyte formation [9], hyperplasia of the affected condyle along with ectopic bones and cartilage in the periarticular region leading to the development of Traumatic TMJ Ankylosis (TTMJA) [10,11].

In our study, we present a pre-clinical mouse model of TMJ articular cartilage defect and investigate a possible tissue engineered therapy for fibrocartilage regeneration. To this end, the authors test the efficacy of a hydrogel-biosilica compound, seeded with Mesenchymal Stem Cells (MSCs) derived from the mice condyle, which was applied on the site of the defect immediately after the injury. The authors hypothesize that the defects treated with the compound will improve the regeneration of the defects compared to the untreated group.

Materials and Methods

Animals

All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Massachusetts General Hospital (Protocol # 2017N000086). Thirty-six C57BL/6 mice (n=36), females, 8-10 weeks old were obtained from Charles River Laboratories (Wilmington, MA). Mice were maintained in a virus and parasitefree barrier facility and exposed to a 12-hour light/dark cycle.

Seeding of biosilica incorporating hydrogel

TMJ condyles of two mice (mice carcasses) were explanted in a sterile manner (n=4). Mesenchymal Stem Cells (MSCs) were harvested, isolated and expanded for 2 weeks to reach approximately 90% confluency. Cells were harvested at a cell viability rate over 90%. Then they were passaged, and we used the second passage of cells for the in vivo study. These steps followed standard MSCs culture method and kept under standard culture conditions as previously described [12]. Thamm et al. (2021; unpublished data) [12] proved that these cells were MSCs using flow cytometry (FACS) analysis.

The gelatine/biosilica-based hydrogel was prepared as follows: 1g of type A gelatin (Sigma-Aldrich, St. Louis, MO), sterilized under UV light for 15 minutes, was mixed with 10mL of sterile PBS in a glass beaker, previously cleaned with distilled water and exposed to UV light. After stirring for 1 hour at 40°C on a hot plate, 0.07g of functionalized biosilica nanoparticles [13,14] were added. The hydrogel containing biosilica nanoparticles was kept stirring for another 1 hour. After receiving a homogenous fluid mixture, the hydrogel was transferred into a sterile tube and placed into a water bath to adjust hydrogel temperature to 37°C. MSCs were added at a ratio of 5x106 cells/mL to hydrogel by gently pipetting up and down to obtain a homogenous cell seeded viscous hydrogel. Finally, 10% w/v hydrogel containing 1x106 cells/mL was aspirated in a sterile syringe and placed in the incubator at 37°C prior application.

Cell viability assay and DAPI (4',6-Diamidin-2-phenylindol) staining

To show cell viability after preparing the compound (hydrogel+biosilica+MSCs) and before delivery at the condylar cartilage defects in mice the following analysis were performed.

Live/dead staining kit (Invitrogen, Carlsbad, CA) was used according to the manufacturer’s instructions. Briefly, after thawing, 1mL of Live Green (Comp. A, 1μM) was added to 1μL of Dead Red (Comp. B) and mixed with Dulbecco’s Modified Eagle Medium (DMEM), to achieve a total of 2mL working solution. Gel droplets were placed into a 6-well-plate, cut into half and were subsequently covered with working for 15 minutes at room temperature in dark. Cells were imaged under fluorescence microscope. After observing cell viability, the same hydrogel specimens were fixed in 10% neutralbuffered formalin for 1 hour, followed by washing and subsequently applying DAPI working solution. Samples were left incubating in dark for 7 minutes at room temperature. DAPI staining was finally evaluated under fluorescence microscope.

Surgical procedure

The anatomy, the surgical technique and the perioperative care of all mice followed a detailed description described by Hakim et al. [15].

Briefly, mice were anesthetized with inhalant isoflurane (Isoflurane USP, Patterson Veterinary, Greeley, CO, USA) and remained under anesthesia during the procedure. The hair of the mouse was removed under sedation and the surgical area was disinfected with Betadine solution (Purdue Pharma, L.P, Stamford, CT, USA). Mice were divided into the following groups: (I) sham group (n=10), the TMJ was opened and closed without creating any defect (S-group); (II) control group (n=10), the TMJ was opened and a linear condylar cartilage defect was created with a no 12 surgical scalpel (CD-group); (III) experimental group (n=10), created defect on condylar cartilage, similar to the previously described manner and hydrogel+biosilica+MSCs (20μl) was directly administered (H-group). After creating the defect in the mandible condyle, the hydrogel was administered over the defect and the TMJ capsule was immediately sutured to keep the hydrogel in place. The joints were not immobilized. Mice were provided soft diet during the first 72 hours post-operatively to minimize any load on their jaws. The mice were checked daily for the first 4 days post-surgery and then every 3 days to assess their health progress and wound healing. All mice of each group were euthanized at either 4 (n=15) or 8 weeks (n=15) after surgery (Figure 1).