Rat Maxillary Molar Tooth Extraction
Animal work was performed according to approved Tufts University IACUC protocols and National Institutes of Health guidelines. Maxillary first and second molars (M1 and M2) were extracted from 6- to 8-week-old female Lewis rats (Taconic, Germantown, NY, USA) under general anesthesia. Rats were administered palliative analgesics via subcutaneous injections twice/day and in the drinking water for 3 days post-implantation and were fed a soft diet.
Periodontal Tissue Harvest and Cell Culture
Extracted molars and associated PDL tissues were pooled and rinsed in sterile Hanks’ balanced salt solution (HBSS). We obtained primary cell digests by incubating tooth roots at 37°C for 1 hr in 50 mL HBSS containing 0.67 mg/mL collagenase type II (Worthington Biochemical Corp., Lakewood, NJ, USA) and 0.3 mg/mL dispase (Roche, Basel, Switzerland). We obtained secondary cell digests by incubating primary digests a second time for 1.5 hrs at 37°C in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA), 20% fetal bovine serum (FBS), containing 2.0 mg/mL collagenase type I (Worthington Biochemical Corp.), and 0.25% trypsin. Primary and secondary cell digests were washed, filtered through a 40-µm cell strainer (BD Falcon, Thermo Fisher Scientific, Pittsburgh, PA, USA), re-suspended in DMEM, 20% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and cultured in humidified 5% CO2 at 37°C with media changes every 2 to 3 days.
Rat PDL Cell Proliferation and Alkaline Phosphatase (ALP) Activity
Cultured rat PDL cell proliferation was monitored in triplicate with the PicoGreen dsDNA quantification kit (Invitrogen) and VICTOR3 fluorimeter (PerkinElmer, Boston, MA, USA). We analyzed the same samples for ALP activity, by mixing 80 µL of each with 100 µL p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA), and 20 µL of 0.5 M 2-amino-2-methyl-1-propanol (Sigma-Aldrich) buffer, for 1 hr at 37°C. Spectroscopic measurements at 405 nm were compared with standard curve serial dilutions of 4-nitrophenol (Sigma-Aldrich).
Colony-forming Unit (CFU) Assays
Five independently harvested rat PDL cell preparations were plated at 2000 cells/T25 flask in DMEM, 20% FBS, cultured for 2 wks, fixed with 10% formalin, and stained with saturated methyl violet (Sigma-Aldrich). Colonies greater than 2 mm in diameter were counted and recorded.
Differentiation Potential of PDL Cells
The differentiation potentials of at least two independently prepared P3 PDL cell preparations were tested as previously described (Zhang et al., 2006
). Briefly, adipogenic differentiation media contained 0.5 mM isobutyl-methylxanthine (IBMX), 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin, and 50 µg/mL of gentamicin. Neurogenic differentiation media contained 10 mM BME, 2% dimethyl sulfoxide (DMSO), and 200 mM butylated hydroxyanisole (BHA). Osteogenic media contained 5 mmol/L of KH2
M dexamethasone, 50 µg/mL of L-ascorbic acid, and 50 µg/mL gentamicin.
Immunohistochemical (IHC) Analyses
Rat PDL cell differentiation was assessed by IHC with primary antibodies anti-BSP (ab52128, Abcam, Cambridge, MA, USA), anti-DSPP (LF-153, Dr. Larry Fisher, NIH), anti-NeuN (MAB377, Millipore, Temecula, CA, USA), anti-STRO-1 (Invitrogen), and osteocalcin (OCN) (Abcam), with the Vectastain ABC staining kit (Vector Laboratories, Burlingame, CA, USA), and fast green counterstain. Negative control samples were assessed with secondary antibody alone.
Quantitative Real-time Polymerase Chain-reaction (qRT-PCR)
RNA was isolated from triplicate, differentiated, and control PDL cell cultures with Trizol reagent (Invitrogen) and reverse-transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR analyses were performed with the ABI Prism 7000 Sequence Detection System (Applied Biosystems), and Assays-on-Demand™ Gene Expression kits, for BSP (Rn01450118_m1), DSPP (Rn02132391_s1), and periostin (Rn01494630_g1), normalized to GAPDH (Rn99999916_s1) and analyzed with the ABI Prism 7000 Sequence Detection Systems version 1.0 software (Applied Biosystems).
Implant Fabrication, Preparation, and Placement
Surface-coated custom implants (2.0 mm x 1.5 mm) were produced by Straumann (Basel, Switzerland) by a Sandblasting Large-grit Acid-etching (SLA) technique to provide macro roughness (Cochran et al., 2002
). Following alveolar bone exposure, the implant site was prepared with a round bur (1.4 mm diameter) and custom-made drill (1.5 mm diameter) with mountable stop (2 mm). P3 cultured rat PDL cells were harvested, re-suspended in Matrigel (BD Sciences), and aliquoted (2 µL, 1 x 106
cells) into fabricated molded wells (1.7 mm diameter). An implant was placed into each well, and the Matrigel mix was hardened at 37°C for 1 hr. The resulting Matrigel coating was about 0.5 mm thick, and could withstand press-fitting. Implant beds were prepared to avoid friction during implantation, and the implants were quite stable. At least two independent cell preparations were used to generate duplicate ‘experimental cell plus Matrigel’ (C+M) and ‘Matrigel alone’ (M) implants. Uncoated titanium implants were used as positive ankylosis controls (Choi, 2000
). Right side cell-seeded and left side negative control implants were placed at the healed M1 extraction site of each rat host.
Implant Harvest and Processing
Harvested rat palates were fixed in 10% buffered formalin ON. Our initial 7 hemi-mandibles with implants were sent for processing at the University of Minnesota Hard Tissue Research Laboratory (Donath and Breuner, 1982
; Rohrer and Schubert, 1992
). These samples were dehydrated, infiltrated with embedding resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany), polymerized at 450 nm, cut to 150 µm (EXAKT Technologies, Oklahoma City, OK, USA), polished to 35-50 µm with sandpaper discs (800-2400 grit, EXAKT micro-grinding system), final-polished with 0.3 µm alumina polishing paste, and stained with Stevenel’s blue and Van Gieson’s picro fuchsin, after epifluorescent and histomorphometric analyses. We chose to process the remaining 14 non-decalcified hemi-maxilla implants at the University of Bern. These samples were embedded in methylmethacrylate, sectioned longitudinally into ~400 µm ground sections by means of a slow-speed diamond saw (Varicut®
VC-50, Leco, Munich, Germany), polished (grain size 4000) to a final thickness of ~150 µm (Knuth-Rotor-3, Struers, Rødovre/Copenhagen, Denmark), and surface-stained with toluidine blue and basic fuchsin (Schenk et al., 1984
Morphological and Histomorphometric Evaluation of Bioengineered PDL Tissues
Implant sections were analyzed at the University of Bern with Leica stereolupe M8® and Dialux® EB light microscopes (Leica, Glattbrugg, Switzerland), 60-mm objective (Nikkor, Nikon), and Nikon DN 100® digital camera (Nikon, Egg, Switzerland). Implant osseointegration was evaluated directly at 160X with an integrative eyepiece for determination of percentage bone-to-implant contact (%BIC). Replicate blinded morphometric analyses were performed for reproducibility. Samples also were evaluated for PDL tissue and blood vessel formation/mm2.