Regenerative Engineering of a Biphasic Patient-Fitted Temporomandibular Joint Condylar Prosthesis

Abstract

Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to implant an acellular regenerative TMJ prosthesis was developed for orthotopic implantation in a pilot goat study. The scaffold incorporated a porous, polycaprolactone-hydroxyapatite (PCL-HAp, 20wt% HAp) 3D printed condyle with a cartilage-matrix-containing hydrogel. A series of material characterizations was used to determine the structure, fluid transport, and mechanical properties of 3D printed PCL-HAp. To promote marrow uptake for cell seeding, a scaffold pore size of 152 ± 68 μm resulted in a whole blood transport initial velocity of 3.7 ± 1.2 mm·s^-1 transported to the full 1 cm height. The Young's modulus of PCL was increased by 67% with the addition of HAp, resulting in a stiffness of 269 ± 20 MPa for etched PCL-HAp. In addition, the bending modulus increased by 2.06-fold with the addition of HAp to 470 MPa for PCL-HAp. The prosthesis design with an integrated hydrogel was compared with unoperated contralateral control and no-hydrogel group in a goat model for 6 months. A guide was used to make the condylectomy cut, and the TMJ disc was preserved. MicroCT assessment of bone suggested variable tissue responses with some regions of bone growth and loss, although more loss may have been exhibited by the hydrogel group than the no-hydrogel group. A benchtop load transmission test suggested that the prosthesis was not shielding load to the underlying bone. Although variable, signs of neocartilage formation were exhibited by Alcian blue and collagen II staining on the anterior, functional surface of the condyle. Overall, this study demonstrated signs of functional TMJ restoration with an acellular prosthesis. There were apparent limitations to continuous, reproducible bone formation, and stratified zonal cartilage regeneration. Future work may refine the prosthesis design for a regenerative TMJ prosthesis amenable to clinical translation.

Keywords: 3D printed; chondrogenic; goat; hydroxyapatite; osteogenic; polycaprolactone.

FIG. 1.

Schematic diagram of the prosthesis and surgical implantation. (A) Two prosthesis designs were orthotopically implanted: (1) A smooth PCL-HAp prosthesis, and (2) a biphasic prosthesis with both PCL-HAp and a chondrogenic hydrogel comprised of PHA, PEGDA, and DVC was syringed onto the interlocking condyle with a recessed surface and UV crosslinked such that the integrated hydrogel was flush with the condyle surface. Both prosthesis designs had a porous internal architecture. (B) Surgical implantation was performed with preauricular and retromandibular incisions. The condyle was resected through the preauricular incision, and then the prosthesis was inserted through the retromandibular incision. (C) To resect the condyle, a cutting guide was inserted and fixed to the mandible. Subsequently, the guide was removed, and the prosthesis was inserted and fixed into position. DVC, devitalized cartilage; PCL-Hap, polycaprolactone-hydroxyapatite; PHA, pentenoate-modified hyaluronic acid; PEGDA, polyethylene glycol.

FIG. 2.

Illustration of the testing setup for load transmission. The upper half of the prosthesis was fixed to a 3D printed ramus-shaped base. A 5 lb_f load was applied to the condyle and the transmitted load was measured with a force-sensitive resistor positioned between the condyle and the base. Following each sample, the force sensitive resistor was calibrated by repeating the test with an unconstrained condyle.

FIG. 3.

Calcium and phosphorous were distributed throughout the 3D printed PCL-HAp. Scanning electron microscopy (SEM) of 3D printed PCL-HAp using energy-dispersive x-ray spectroscopy exhibited homogeneously distributed calcium and phosphorous. Scale bar for left-hand image of prosthesis was 10 mm. Scale bars for SEM and EDS were 1 μm. EDS, energy-dispersive X-ray spectroscopy; Ca, calcium; P, phosphorus.

FIG. 4.

Uniaxial tension of 3D printed PCL-HAp. Adding HAp significantly enhanced PCL's mechanical performance. (A) Illustration of test following ASTM D1708. (B) Representative stress/strain plot where letters C–H refer to the parameters reported in the following panels. (C) The addition of HAp significantly enhanced PCL stiffness, but not after NaOH etching compared to nonetched PCL-HAp. (D) Strain energy decreased with the addition of HAp, but not after NaOH etching compared to nonetched PCL-HAp. (E) The ultimate tensile strengths did not differ among groups. (F) Ultimate strains decreased with the addition of HAp, but not after NaOH etching compared to nonetched PCL-HAp. (G) Similar yield stresses were exhibited across the different groups. (H) The yield strain decreased with the addition of HAp to PCL, but not after NaOH etching compared to nonetched PCL-HAp. n = 5–6. *p < 0.05. All data are mean ± standard deviation. HAp was 20% wt.

FIG. 5.

Three-point bend test of 3D printed composites following ASTM D790. The addition of HAp increased PCL flexural stiffness, but the reduction in stiffness after NaOH etching (ePCL-HAp) was not different from nonetched PCL-HAp. n = 6–7. *p < 0.05. All data are mean ± standard deviation. HAp was 20 wt%.

FIG. 6.

Evaluation of the load transmitted through the condyle to the underlying bone geometry. (A) As the collar region of the prosthesis was reduced in thickness from ∼5 mm (thick) to 3 mm (thin), the transmitted load increased by over 10%. (B) Thick and thin collar prostheses. %Load transmitted = F_transmitted/F_applied * 100%. ^#Thick specimen was the design parameter selected for the prostheses implanted in the pilot animal study. The pilot study used full-length prostheses that extended to the angle of the mandible. Scale bar: 10 mm. *p < 0.05. n = 4. Data are mean ± standard deviation.

FIG. 7.

Three-dimensional printed PCL-HAp composite scaffold exhibited capillary rise of whole blood. (A) The rates decreased as pore sizes increased (150, 350, and 550 μm). Negligible capillary rise was exhibited by scaffolds that were not etched (green plot with arrow). A line was fit to the initial data points to calculate each group's initial velocities. (B) The etched group initial velocities decreased by 31% and 77% for 350 and 550 μm groups from 3.7 ± 1.2 mm·s⁻¹ for the 150 μm group. Negligible capillary rise was detected in the nonetched groups (gray bars). Differences were exhibited among all groups, except within nonetched groups (gray bars) and the etched 150 and 350 groups. (C) The prosthesis was designed with a 150 μm porous structure inside the condylar head. Representative specimen illustrated digital measurement of whole blood height (green “Xs”). ^#Denotes 150 μm pore size used in the animal study. Data points are ensemble averages, bands are standard deviations. p < 0.05. n = 7.

FIG. 8.

Assessment of bone with MicroCT data prepared from each subject 6 months after implantation. Gross images were captured from the tissue's superior viewpoint with the camera pointed from the superior perspective. Renderings for each case were displayed with the view direction listed across the top row (superior, anterior, posterior, medial, and lateral). In each case, bone growth was exhibited around the prosthesis condyle, but not inside the condyle's porous region. Bone resorption was observed in all cases with more pronounced bone loss in groups with the hydrogel (D, and E). The control group is shown as the mirror image of the left condyle from subject C to facilitate visual comparison with the experimental group right condyles. In the CT images, the prosthesis, ramus bone, and fossa bone were labeled as #, @, and ^, respectively. The prosthesis was displayed as white, and bone as brown in the renderings. Dashed line in ‘superior’ column represents plane of CT image in right-most column. The approximate distance between the prosthesis collar and bone was shown with a red line superimposed on the CT sections. The approximate lengths were 6.0, 5.6, and 2.6 mm for nonhydrogel groups A, B, and C, and 7.2, 9.2 mm for groups D and E, respectively. MicroCT imaging volume was focused on the condylar region, although the prostheses extended down to the angle of the mandible. Scale bar: 10 mm.

FIG. 9.

Assessment of neocartilage and bone formation with histology and immunohistochemistry from selected regions of sagittal condyle sections. Note in the healthy control tissue the deeper hyaline-like cartilage layer (black arrows) below the overlying proliferative and superior fibrous zones. Cases C (without hydrogel) and D (with hydrogel) exhibited evidence of cartilage-like soft tissue regeneration. Alcian blue staining was exhibited by the control mandibular condylar cartilage's deep layer, and in subjects A, B, C, and D with the richest experimental tissue staining in C and D. Collagen II staining was exhibited by the control specimen's deep layer, in addition to subjects C and D on the anterior, articulating surfaces. Subject B exhibited faint collagen II staining in the pericellular matrix. Generally, high collagen II staining was localized to the pericellular matrix. Specimens A and E exhibited darker CD4 staining for helper T cells than the other cases, suggesting a variable innate immune response. Little to no CD4 staining was exhibited by the contralateral control condyle. The disc fused to the surface of condyle D shown with *. Gross images presented the tissue sections before slide mounting. Illustration of ramus and condylar prosthesis (left panel) with sagittal plane was provided for orientation purposes. Dashed lines represent prosthesis border. Scale bars: 3 mm for H&E, 50 μm for insets, and 200 μm (lower right) for all other images. H&E, hematoxylin and eosin.

FIG. 10.

Assessment of TMJ disc structure with histology. Disc outcomes were variable from nearly pristine to perforated. MicroCT after tissue resection illustrates approximate disc location relative to condyle and fossa. Gross images exhibit changing sizes among disc tissues. Subjects A and E exhibited granulation tissue, with some remaining discernable disc tissue in subject E (arrow). Subject C disc tissues matched the histological structure of the control, although the aspect ratio appears to have changed to a shorter medial-lateral and longer anterior-posterior than control disc. In subject D, the disc fused to the condyle; hence, there were no data. Alcian blue staining was positive in a distinct band in the control disc (*). Alcian blue staining persisted in B and C, although was largely absent in A and E. The overall gross morphology score was highest in subject C. Details regarding scoring information can be found in Supplementary Table. Orientations were medial (M), lateral (L), anterior (A), and posterior (P) for the gross images; and superior (S), and inferior (I) for histology images. Control displayed as mirror image for comparison with experimental data contralateral side of jaw. Dashed lines denote sectioning. H&E staining. Scale bars are 10 mm for gross images, 50 μm for H&E magnified column, and 3 mm for remaining images (lower right). TMJ, temporomandibular joint.