Experimental Formation of Periodontal Structure Around Titanium Implants Utilizing Bone Marrow Mesenchymal Stem Cells: A Pilot Study
Tissue engineering in the head and neck area, presents numerous advantages. One of the most remarkable advantages is that regeneration of only a small amount of tissue can be highly beneficial to the patient, particularly in the field of periodontal tissue regeneration. For decades, successful osseointegration has provided thousands of restorations that maintain normal function. With the increasing need to utilize dental implants for growing patients and enhance their function to simulate normal tooth physiology and proprioception, there appears to be an urgent need for the concept of periodontal tissue regeneration around dental implants. In the present work, 5 goats were used for immediate implant placement post canine teeth extraction. Each goat received 2 implant fixtures; the control side received a porous hollow root-form poly (DL-Lactide-co-Glycolide) scaffold around the titanium fixture, and the experimental side received the same scaffold but seeded with autogenous bone marrow–derived mesenchymal stem cells. One animal was killed 10 days postoperatively, and the others were killed after 1 month. The results showed that on the experimental side, periodontal-like tissue with newly formed bone was demonstrated both at 10 days and after 1 month, while the control specimens showed early signs of connective tissue regeneration around the titanium fixture at 10 days, but was not shown in the 1 month specimens. It can be concluded that undifferentiated mesenchymal stem cells were capable of differentiating to provide the 3 critical tissues required for periodontal tissue regeneration: cementum, bone, and periodontal ligament. This work may provide a new approach for periodontal tissue regeneration.Abstract

Prototype of designed Teflon mold used to produce hollow porous scaffold around the dental implant screw. (a) The dimensions of the insert and the tube. (b) The insert, the hollow tube, and the solid cylinder with a flat surface. (c) The three parts as they are used (left side of photo) and the hollow Teflon tube, with insert inside showing the space to be occupied later by the DL-Lactide-co-Glycolide (PLG) scaffold (right side of photo). (d and e) The PLG scaffold at the end of the insert as it comes out after molding.

Isolation of bone marrow from goat. (a and b) An 8 mm skin incision was made in the thigh region to expose the bone, then a hole was drilled in the bone of the femur using a micromotor. (c and d) The marrow was collected from the femur with the use of a 20 mL syringe with a 14 gauge cannula.

Implantation of endosseous titanium dental fixture and DL-Lactide-co-Glycolide (PLG) scaffold/cells in fresh extraction socket of the goat canine tooth. (a) Schematic drawing of goat mandible indicating the area of the experiment. (b and c) The extraction procedure. (d and e) The procedure of fixture implantation. (f) Insertion of PLG scaffold/cells construct around the fixture.


Characterized DL-Lactide-co-Glycolide (PLG) degraded samples under dynamic loading. (a) PLG scaffold dimensions, which include 5 mm outer diameters, a 4 mm inner diameter, and a 5 mm length. (b) Scanning electron microscopy (SEM) of PLG scaffold shows porosity and voids in the polymer scaffold. SEM ×35. (c) PLG scaffold 1 week after degradation. SEM ×35. (d) One week degraded scaffold after being exposed to dynamic compression load up to 4000 Pa. SEM ×200.

Culturing goat bone marrow derived mesenchymal stem cells. (a) Day 6 ×20. (b) Day 10 ×20. (c) Day 12 ×20. (d) Day 18 ×20.

Seeding goat bone marrow derived mesenchymal stem cells onto porous DL-Lactide-co-Glycolide (PLG) scaffold. (a) PLG seeded scaffold after 2 days under inverted light microscope. (b) Photograph for the seeded scaffold before implantation. (c) Scanning electron microscopy (SEM) view for the porous surface of unseeded scaffold. SEM ×2000. (d) SEM view for the seeded porous surface scaffold. SEM ×2000.

Radiographic views of the 10 days retrieved bony specimen of goat mandible. (a) Overall radiographic view of the whole mandible, showing both control side (right side) and experimental side (left side) (original magnification ×1). (b and c) Radiographic views of the goat mandible (side view) showing control side at (b) and experimental side at (c) (original magnification ×1). Note the similarities of the interfaces: Tooth/surrounding tissue and implant/surrounding tissue.

Histologic evaluation for the 10 day specimen at the control side. (a) Overall implant fixture with the surrounding peri-implant tissue showing the surface of the implant fixture, newly formed bone around it, and part of the bony socket wall (original magnification ×10). (b) Connective tissue fibers extending from the socket wall through the openings in the newly formed bone toward the implant serration (original magnification ×40). (c and d) Larger magnification of the area at (b), showing the bundles of connective tissue fibers extending with intimate contact to the new bone surface (arrows) (original magnification ×100).

Histologic evaluation for the 10 day specimen at the control side. (a) Overall implant fixture with the surrounding peri-implant tissue shows the surface of the implant fixture, newly formed bone around it, and part of the inner bony socket wall (toward midline of the jaw) (original magnification ×10). (b) Newly formed bone adapted directly to the metal surface of the implant (osseointegrated) (original magnification ×40). (c and d) Newly formed bone extending toward implant serration comprised of woven bone and parallel fibers, while the center of the bone shows varying amounts of lamellar bone (original magnification ×40 at [c] and ×100 at [d]).

Histologic evaluation of the 10 day specimen at the experimental side. (a) Overall implant fixture with the surrounding peri-implant tissue shows the newly formed, almost continuous, bony trabeculae all around the implant fixture (original magnification ×10). (b) The bony wall of the extraction socket and the remaining periodontal tissue extending from the socket wall through an opening between the newly formed trabeculae toward the implant fixture (original magnification ×40). (c) Larger magnification for the top part of the previous photo shows the remaining periodontal tissue extending from the socket wall to completely surround the newly formed woven bone and the connective tissue fibers extending toward the implant fixture (original magnification ×40). (d) Larger magnification for the bottom part of the photo at (b) shows the periodontal tissue that extends from the socket wall to completely surround the newly formed bone. Bone remodeling is obvious, the arrows indicate the presence of osteoclasts at the outer surface of the newly formed bone away from the implant fixture, and the arrowheads indicate the newly formed woven bone. The same photo shows connective tissue fiber bundles tightly attached to the surface of the newly formed bone. The surface of the titanium fixture exhibits attachment of the circular fibers that extend toward the newly woven bone (original magnification ×100).

Histologic evaluation of the 10 day specimen at the experimental side implant/tissue interface. (a and c) Preservation of the original width of the periodontal ligament between the titanium fixture and the newly formed bone. Although the direction of the fibers was not organized in the coronal part, they seem parallel to the titanium surface, then change direction to horizontal toward the middle of the fixture, to run from the surface of the fixture to be inserted into the newly formed bone (original magnification ×40). (b and d) Higher magnification of the previous photos demonstrating newly formed bone and bundles of collagen fibers that are inserted into the titanium surface (original magnification ×100).

Histologic evaluation of collagen fiber bundles in the 10 day specimen of the experimental side implant/tissue interface, coronal area. (a) Histologic section at the coronal part of the implant/tissue interface shows bundles of periodontal ligament fibers running parallel to the implant surface, and newly formed bone inserted into both surfaces (original magnification ×40). (b and c) Larger magnification shows the highly cellular network of periodontal-like fibers filling the space between implant and bone while inserted into the surface of the newly formed bone from one side and of the newly formed cementum matrix on the implant surface on the other side (original magnification ×100). (d) Large magnification shows the periodontal-like fibers as they are inserted into the newly formed cementum matrix on the titanium surface and active newly formed woven bone surface (original magnification ×400).

Formation of cellular cementum-like layer at the surface of the titanium screw at the experimental side (10 days specimen). (a and b) Continuity of cementum-like layer with even thickness deposited on the titanium surface (original magnification ×400). (c) Even thickness of cementum-like layer with extrinsic fibers similar to Sharpey's fibers (original magnification ×200). (d) Higher magnification for the titanium surface shows the deposited cellular cementum and the extracellular matrix with almost even thickness (original magnification ×400).

Histologic evaluation for the 4 week specimen at the control side. (a) Healed bony socket at the control side after removal of titanium fixture (original magnification ×10). (b) The side wall of the bony socket where bone trabeculae extend from the coronal part of original socket wall toward the implant fixture space (original magnification ×40). (c) Larger magnification for the socket wall at the side with few bony spicules extending from socket wall toward the implant fixture space (original magnification ×100).

Histologic evaluation for the 4 week specimen at the experimental side. (a) Macroscopic view for the bony specimen before implant fixture removal. (b) Longitudinal section at the place of the implant fixture shows the empty socket after implant retrieval with double bony walls; the outside is the original socket wall and the inside is the new bony wall (original magnification ×10). (c) Larger magnification for the socket wall shows the newly formed bony trabeculae parallel to the socket wall and newly formed bony spicules toward the center of the socket at larger magnification for the square area at (b). (d) De novo regenerated bone extends toward the center of the socket. These fine spicules are lined by osteoblast cells (original magnification ×400).

Histologic evaluation for the regenerated periodontal-like tissue in the space surrounded by the inside bony wall experimental side at 4 weeks. (a) Overall view for the peri-implant tissue area surrounded by the inside bony wall of bone, showing epithelium coverage, de novo trabecular bone lined by organized bony spicules, and a band of periodontal-like tissue (from outside toward implant) (original magnification ×10). (b) Section showing the superficial epithelium over the socket area that covers the titanium implant fixture (original magnification ×40). (c and d) Section (part of the side wall) showing bony spicules with bundles of connective tissue fibers extending between the previously mentioned newly formed bony spicules toward the space where the implant fixture existed (original magnification: [c] ×100, [d] ×400). (e) Section representing the bottom area in the peri-implant tissue located at the fundus of the socket; de novo bony spicules are apparent, and collagen bundles are running from one side to the other in the socket area, and in between the bony spicules. At larger magnification (f), highly cellular bundles of connective tissue fibers are very obvious to surround the bony spicules (original magnification: [e] ×100, [f] ×400).

Scanning electron microscope for the titanium fixture retrieved from the 4 week specimen at the experimental side. (a) Overall view for the retrieved titanium fixture (SEM ×25). (b) Area between serrations 2 and 3 showing part of the peri-implant tissue attached to the surface of the serration covered with densely packed collagen fibers similar to natural cementum; typical perforated-like bone structure appears at the outer layer of the peri-implant tissue (SEM ×140). (c and d) Area between serrations 3 and 4 showing periodontal-like tissue with numerous bundles of collagen fibers attached, while many were cut during fixture removal (SEM: [c] ×170, [d] with larger magnification for 1 bundle; original magnification ×1500). (e and f) Area between serrations 4 and 5 showing serrations covered with cementum-like structure and periodontal-like tissue with obvious numerous collagen bundles of fibers attached to the cementum-like structure covering the fixture serration. Although the densely packed collagen fibers of the cementum are running parallel to the implant, the collagen bundles of periodontal tissue are attached to the cementum and are growing perpendicular (SEM: [e] ×250, [f] ×1500).
