Attenuation of myogenic orofacial nociception and mechanical hypersensitivity by viral mediated enkephalin overproduction in male and female rats.

BACKGROUND: Clinical studies have tested the use of an engineered herpes virus to treat pain. We hypothesized that subcutaneous injections of an engineered herpes virus that expresses enkephalin would attenuate orofacial nociception and hypersensitivity in male and female rats by a central mechanism. METHODS: Herpes virus was injected subcutaneously around the mouth of male and female rats seventy-two hours before ligatures were placed on the masseter tendon, control treatment groups received either no virus or no ligature. Enkephalin expression was measured and von Frey filament testing and meal duration were utilized to measure mechanical hypersensitivity and the nociceptive response, respectively. Naloxone or naloxone methiodide was administered to rats injected with the enkephalin expressing virus to test if enkephalin was acting peripherally or centrally. RESULTS: Ligature significantly lengthened meal duration and reduced the threshold to von Frey filaments for 18 days. Infection with the enkephalin transgene significantly decreased this response for at least 11 days but only in male rats. Virus injection significantly increased expression of enkephalin in the mental nerve that innervates the mouth region, the trigeminal ganglia and the trigeminal nucleus caudalis but no increase was observed in the masseter nerve after virus injection. Naloxone but not naloxone methiodide reversed the response to the enkephaline expressing virus. CONCLUSIONS: The data suggests that sex should be a considered when using this virus and that viral transfection of the mental nerve with an enkephalin transgene can reduce nociception and hypersensitivity through a central mechanism.

Intracranial calcification in Fam20c-deficient mice recapitulates human Raine syndrome.

FAM20C (family with sequence similarity 20-member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. FAM20C loss-of-function mutations cause Raine syndrome in humans, characterized by generalized osteosclerosis, distinctive craniofacial dysmorphism, along with extensive intracranial calcification. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets. In this study, we examined the expression of Fam20c in the mouse brain and investigated brain calcification in Fam20c-deficient mice. Reverse transcription polymerase chain reaction (RT-PCR), Western-blotting and in situ hybridization analyses demonstrated the broad expression of Fam20c in the mouse brain tissue. X-ray and histological analyses showed that the global deletion of Fam20c (mediated by Sox2-cre) resulted in brain calcification in mice after postnatal 3 months and that the calcifications were bilaterally distributed within the brain. There was mild perifocal microgliosis as well as astrogliosis around calcospherites. The calcifications were first observed in the thalamus, and later in the forebrain and hindbrain. Furthermore, brain-specific deletion (mediated by Nestin-cre) of Fam20c in mice also led to cerebral calcification at an older age (postnatal 6 months), but no obvious skeletal or dental defects. Our results suggest that the local loss of FAM20C function in the brain may directly account for intracranial calcification. We propose that FAM20C plays an essential role in maintaining normal brain homeostasis and preventing ectopic brain calcification.

Selective blockade of the rat brain aqueduct with thermogelling hydrogel nanoparticle dispersion.

Experimental methods targeting molecules or drugs to specific neuronal tissue(s) can be important in determining function. In this study we focused on blockade of the small channel or aqueduct connecting the third and fourth ventricles of the rat brain. A cannula was placed into the aqueduct between the third and fourth ventricle. A second cannula was placed into the third or fourth ventricle. An aqueous dispersion of hydrogel nanoparticles, that maintains a liquid state at temperatures below 33 degrees C and solidifies near body temperature (35 degrees C), was infused into the aqueduct. Two interpenetrating polymer networks (IPN) of hydrogel nanoparticles with polymer concentrations at 2% by weight and 3% by weight were separately infused into the aqueduct to block cerebrospinal fluid (CSF) flow. Following infusion of hydrogel CSF was isolated to a particular ventricle as shown by the lack of dye movement between the ventricles. In addition, stress hormone, corticosterone, feeding behavior and blood glucose levels were measured. Results show upon reaching the aqueduct the hydrogel dispersion solidified and restricted the flow of CSF. A higher concentration of dispersion (3% wt.) was more effective in blocking the aqueduct and isolating the third from the fourth ventricle. Over the period of measurement, infusion of the dispersion had no measurable detrimental physiological effects on the animal. We conclude that isolation of ventricles in the brain can be completed for 48-h by using dispersions of hydrogel nanoparticles and the effects of drugs on certain brain tissues can be determined with this method.

Capping a pulpotomy with calcium aluminosilicate cement: comparison to mineral trioxide aggregates.

INTRODUCTION: Calcium aluminate cements have shown little affinity for bacterial growth, low toxicity, and immunogenicity when used as a restoration material, but calcium aluminate cements have not been tested in vivo in pulpotomy procedures. METHODS: To address this question, a calcium aluminosilicate cement (Quick-Set) was tested along with 2 mineral trioxide aggregates, ProRoot MTA and MTA Plus. These cements were used as a capping agent after pulpotomy. Control rats had no pulpotomy, or the pulpotomy was not capped. Proinflammatory cytokines interleukin (IL)-1ß and IL-1α were measured, and histology was performed at 30 and 60 days after capping. The nociceptive response was determined by measuring the lengthening of the rat's meal duration. RESULTS: and CONCLUSIONS: IL-1ß and IL-1α concentrations were reduced in the capped teeth, but no differences were observed among the 3 cements. Dentinal bridging could be detected at both 30 and 60 days with each of the 3 cements, and the pulps were still vital 60 days after capping. Meal duration significantly shortened after placement of the 3 different cements, indicating a nociceptive response, but there were no differences among the materials. Calcium aluminosilicate cement had similar properties to mineral trioxide aggregates and is a viable option for pulpotomy procedures.

Intra-articular controlled release of anti-inflammatory siRNA with biodegradable polymer microparticles ameliorates temporomandibular joint inflammation.

We investigated the in vivo therapeutic efficacy of an intra-articular controlled release system consisting of biodegradable poly(dl-lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating anti-inflammatory small interfering RNA (siRNA), together with branched poly(ethylenimine) (PEI) as a transfecting agent, in a rat model of painful temporomandibular joint (TMJ) inflammation. The in vivo effects of PLGA MP dose and siRNA-PEI polyplex delivery were examined via non-invasive meal pattern analysis and by quantifying the protein level of the siRNA target as well as of several downstream inflammatory cytokines. Controlled release of siRNA-PEI from PLGA MPs significantly reduced inflammation-induced changes in meal patterns compared to untreated rats with inflamed TMJs. These changes correlated to decreases in tissue-level protein expression of the siRNA target to 20-50% of the amount present in the corresponding control groups. Similar reductions were also observed in the expression of downstream inflammatory cytokines, e.g. interleukin-6, whose tissue levels in the siRNA-PEI PLGA MP groups were 50% of the values for the corresponding controls. This intra-articular sustained release system has significant implications for the treatment of severe TMJ pain, and also has the potential to be readily adapted and applied to mitigate painful, chronic inflammation in a variety of conditions.

Multipotent adult progenitor cells acquire periodontal ligament characteristics in vivo.

Mesenchymal stem cells (MSC) and multipotent adult progenitor cells (MAPC) copurify from the bone marrow. Past studies have shown that bone marrow-derived cells transplanted into the periodontium obtain characteristics of several periodontal cells types, such as alveolar bone, periodontal fibroblasts, and cementoblasts. Bone marrow-derived cell populations are heterogeneous mixtures of different cell types. Characterization of each individual cell type in the bone marrow has not been completed. In this study, the MAPC were isolated from the bone marrow of 3-week-old rats by selecting attached cells that did not express leukocyte (CD45), erythroid (HIS49), or the MSC marker CD44. DiI-labeled male MAPC were injected into the periodontal ligament (PDL) of female adult rats. After 3 weeks, the morphology of the DiI-labeled cells was determined. Periodontal tissues sections that included alveolar bone, PDL, cementum, and dentin were also immunostained for collagen I and III after 3 days, 3 weeks, and 6 weeks following injection of male MAPC into the female PDL. Male and female cells were differentiated by fluorescent in situ hybridization tagging of the X and Y chromosomes. Our results show that DiI-labeled MAPC were spherical in shape before injection but after 3 weeks a portion of the injected MAPC obtained the characteristic spindle shape of PDL cells with an orientation parallel to the surrounding PDL cells. Consistent with the DiI results, male MAPC had a spherical shape on immunostained sections 3 days after injection and expressed collagen I and III at significantly reduced levels as compared with the endogenous female PDL. Six weeks after injection, the MAPC expressed both collagen I and collagen III at levels similar to the endogenous surrounding PDL and obtain a morphology characteristic of PDL. In conclusion, MAPC obtain PDL-like characteristics over a 6-week period after injection into the PDL.

Integrin mediated attachment of periodontal ligament to titanium surfaces.

OBJECTIVE: Reducing the force between the implant and the bone by recapitulating a similar matrix has the potential to reduce implant failure. To begin to pursue the goal of creating a periodontal ligament interface between a dental implant and bone, the mechanism of cellular attachment to dental implant surfaces must be characterized. METHODS: In this study we examined the role of integrin receptors in the attachment of periodontal ligament fibroblasts to titanium surfaces utilized on dental implants; those surfaces included smooth polished titanium, acid pickled titanium, ground titanium, sandblasted and acid etched titanium, non-oxidized titanium that has been sandblasted and acid etched, hydroxyapatite coated titanium, titanium plasma sprayed or uncoated titanium. For these studies integrin mediated fibroblast attachment was blocked by the integrin blocking peptide GRGDSP or anti-integrin beta1 antibody or a combination of the two. Quantitation of periodontal ligament fibroblast attachment was completed by counting cells on the various implant surfaces after culturing in vitro for 24h with and without the integrin receptor blockers. RESULTS: Antibody and peptide treatment significantly reduced the number of fibroblasts cells attached to the various implant surfaces but this effect varied significantly depending on the surface. Moreover, increased levels of peptide further decreased fibroblasts attachment in a dose dependent manner. SIGNIFICANCE: Blocking studies suggest first, that integrin receptors function in periodontal ligament attachment to titanium surfaces and second, that different integrin subunits are important in attachment to a particular surface.

Survival of human periodontal ligament cells in media proposed for transport of avulsed teeth.

Many solutions have been examined as possible storage media for avulsed teeth. In this report, human periodontal ligament (PDL) cells were exposed for 1 h to culture medium, milk, Hanks Balanced Salt Solution (HBSS), Soft Wear, Opti Free, and Solo Care contact lens solutions, Gatorade, and tap water, at room temperature and on ice. The number of viable cells was counted using the trypan blue exclusion technique, immediately after exposure (0 h) and at 24 and 48 h, to test the proliferative capacity of the cells after treatment. The results indicated that a significantly higher number of cells survived and proliferated when the exposures were performed at 0 degrees C. Water had a detrimental effect on the cells, whereas culture medium and HBSS preserved significantly more viable cells than the other experimental solutions. Within the parameters of this study, it appears that HBSS is the optimal storage medium for avulsed teeth. Low-fat milk could serve as an alternative if ice is available. Contact lens solutions or Gatorade on ice could serve as short-term (1 h) storage media if the other solutions are not readily available.