Evaluation of bone regeneration potential of dental follicle stem cells for treatment of craniofacial defects.

BACKGROUND AIMS: Stem cell-based tissue regeneration offers potential for treatment of craniofacial bone defects. The dental follicle, a loose connective tissue surrounding the unerupted tooth, has been shown to contain progenitor/stem cells. Dental follicle stem cells (DFSCs) have strong osteogenesis capability, which makes them suitable for repairing skeletal defects. The objective of this study was to evaluate bone regeneration capability of DFSCs loaded into polycaprolactone (PCL) scaffold for treatment of craniofacial defects. METHODS: DFSCs were isolated from the first mandibular molars of postnatal Sprague-Dawley rats and seeded into the PCL scaffold. Cell attachment and cell viability on the scaffold were examined with the use of scanning electron microscopy and alamar blue reduction assay. For in vivo transplantation, critical-size defects were created on the skulls of 5-month-old immunocompetent rats, and the cell-scaffold constructs were transplanted into the defects. RESULTS: Skulls were collected at 4 and 8 weeks after transplantation, and bone regeneration in the defects was evaluated with the use of micro-computed tomography and histological analysis. Scanning electron microscopy and Alamar blue assay demonstrated attachment and proliferation of DFSCs in the PCL scaffold. Bone regeneration was observed in the defects treated with DFSC transplantation but not in the controls without DFSC transplant. Transplanting DFSC-PCL with or without osteogenic induction before transplantation achieved approximately 50% bone regeneration at 8 weeks. Formation of woven bone was observed in the DFSC-PCL treatment group. Similar results were seen when osteogenic-induced DFSC-PCL was transplanted to the critical-size defects. CONCLUSIONS: This study demonstrated that transplantation of DFSCs seeded into PCL scaffolds can be used to repair craniofacial defects.

Optimizing the Glass Bead Sterilization Protocol Focusing on Removal of Organic and Bacterial Intraoperative Contamination.

Validated glass bead sterilization protocols to effectively sterilize rodent surgical instruments after bacterial exposure (for example, cecal contamination) are lacking. To refine current approaches, we added either a multienzyme detergent, neutral pH detergent, or chlorhexidine scrub step before glass bead sterilization of forceps or needle drivers exposed to cecal contents. We exposed sets of forceps and needle drivers to cecal contents, which were then air dried for 3 min. Immediately after, the instruments were wiped several times with a clean, dry paper towel. The contaminated tips were soaked in either a multienzyme or neutral pH detergent (t = 5 min), chlorhexidine scrub (t = 2 min), or no pretreatment solution. To further increase debris removal, instruments (from all groups) were brushed using a clean toothbrush. The nonpretreatment instruments were briefly soaked in saline before brushing. After being rinsed with sterile water, all instruments were exposed to a glass bead sterilizer for 60 s at 500 °F (260 °C). Sets were then swabbed for bacterial culturing. Swabs were plated onto either sheep blood agar (n = 23) or chocolate agar (n = 20) for aerobic culturing or Brucella agar (n = 20) for anaerobic culturing. A subset of instruments was sampled to determine organic material presence after treatment using an ATP luminometer (n = 21). Multiple agar types and bioluminescence were used to more deeply evaluate tool sterility and to differentiate the relative effectiveness of each protocol. From the saline group, only one pair of forceps yielded growth on Brucella agar, and 2 pairs yielded growth on chocolate agar. No other bacterial growth was observed. The use of a pretreatment agent also lowered overall organic contamination levels in needle drivers compared with using only saline. These results indicate that brushing instruments to mechanically remove debris from instruments is paramount to ensure sterility. However, a best practice would be to also use one of the pretreatment options used in this study.

A spontaneous compound odontoma in an adult Sprague Dawley rat (Rattus norvegicus).

Reports of compound odontomas in rats are very rare. A 14-month-old adult male Sprague Dawley rat was found to have a hard mass associated with the caudal aspect of the left mandible. After 2 weeks of observation, the rat was euthanized due to the mass growing significantly in size and the rat losing >20% of its body weight. Grossly, the mass was well-circumscribed, 3.7 × 3 × 1.2 cm, hard and heterogeneously coloured white, tan and red. The mass was restricted to the mandibular bone and did not involve surrounding subcutaneous tissue. On cut surface, the mass was a similar colour and brittle. Histologically, there were numerous proto-teeth embedded in ossified stroma. Each proto-tooth had a central mesenchyme pulp surrounded by columnar odontoblasts and dentine matrix. The dentine was often bordered by enamel matrix, which was occasionally bounded by ameloblasts. These histological findings were consistent with a compound odontoma. This is the first report of a spontaneous compound odontoma in the caudal mandible of a rat.

Tissue-engineered autologous grafts for facial bone reconstruction.

Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care-the use of bone harvested from another region in the body-has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, native bovine bone matrix, and a perfusion bioreactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyle unit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatán minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material and crafted it into an anatomically correct shape using image-guided micromilling to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either nonseeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering.

Bupivacaine mandibular nerve block affects intraoperative blood pressure and heart rate in a Yucatan miniature swine mandibular condylectomy model: a pilot study.

PURPOSE/AIM: The primary objective was to evaluate the effect of a bupivacaine mandibular nerve block on intraoperative blood pressure (BP) and heart rate (HR) in response to surgical stimulation and the need for systemic analgesics postoperatively. We hypothesized that a mandibular nerve block would decrease the need for systemic analgesics both intraoperatively and postoperatively. MATERIALS AND METHODS: Fourteen adult male Yucatan pigs were purchased. Pigs were chemically restrained with ketamine, midazolam, and dexmedetomidine and anesthesia was maintained with isoflurane inhalant anesthesia. Pigs were randomized to receive a mandibular block with either bupivacaine (bupivacaine group) or saline (control group). A nerve stimulator was used for administration of the block with observation of masseter muscle twitch to indicate the injection site. Invasive BP and HR were measured with the aid of an arterial catheter in eight pigs. A rescue analgesic protocol consisting of fentanyl and lidocaine was administered if HR or BP values increased 20% from baseline. Postoperative pain was quantified with a customized ethogram. HR and BP were evaluated at base line, pre-rescue, 10 and 20 min post-rescue. RESULTS: Pre-rescue mean BP was significantly increased (p = .001) for the bupivacaine group. Mean intraoperative HR was significantly lower (p = .044) in the bupivacaine versus saline group. All other parameters were not significant. CONCLUSION: Addition of a mandibular nerve block to the anesthetic regimen in the miniature pig condylectomy model may improve variations in intraoperative BP and HR. This study establishes the foundation for future studies with larger animal numbers to confirm these preliminary findings.

Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs.

BACKGROUND: Strains of Dirofilaria immitis suspected of lack of efficacy (LOE) to macrocyclic lactone (ML) preventive drugs have been increasingly reported in dogs by practicing veterinarians since 2005 in the Lower Mississippi Delta region. If proven, and not controlled in the early stages, the emergence of ML drug resistance threatens to become a widespread problem in the US that may limit the effectiveness of current preventive drug treatment methods. METHODS: To validate practice reports, a statewide survey of Louisiana veterinarians was done to define the extent of the problem and identify focal 'hotspots' of reported ML LOEs using Geographic Information Systems (GIS) methods. The present study then utilized microfilariae (Mf) from two canine field cases from different state locations that fit criteria for a high index of suspicion of LOE against heartworms by ML drugs. Blood containing Mf from the canine field cases was used to infect and produce L3 in Aedes aegypti for experimental infection of two groups of dogs, each of which contained two laboratory dogs, one treated with prophylactic ivermectin (12 µg/kg) monthly for 6 months at twice the label dose (6 µg/kg), and one untreated control. RESULTS: Both treated and untreated dogs from Group I and Group II developed patent D. immitis infections by 218 DPI and 189 DPI, respectively, as evidenced by a positive occult heartworm antigen test and microfilaremia by the Knott's test. Mf counts gradually increased post-patency in test and control dogs. Infective larvae raised from microfilariae from the treated Group I dog were used to successfully establish a second generation isolate, confirming heritability of resistance in the face of a monthly ivermectin challenge dose of 24 µg/kg, given monthly for 3 months. CONCLUSIONS: These experimental infection studies provide in vivo evidence of the existence of ML drug resistance in dogs infected by D. immitis L3 from suspect field LOE cases in the Lower Mississippi Delta. Results encourage further work on mechanisms underlying the emergence of ML resistance in D. immitis and development of evidence-based resistance management strategies for heartworm preventives in order to extend the useful life of current drugs.