Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model.

OBJECTIVE: The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. METHODS: Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. RESULTS: Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. CONCLUSION: The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair.

Transverse cervical nerve: implications for dental anesthesia.

The inferior alveolar nerve block (IANB) has the highest failure incidence of any dental anesthetic technique. Many authors have outlined potential reasons for these failures in permanent lower molars, including accessory innervations from the mylohyoid and mental foramen. However, the potential accessory innervation of posterior mandibular teeth from the transverse cervical nerve (TCN), a branch of ventral rami from the C2-C3 spinal nerves from the cervical plexus (CP), has been difficult to assess as a result of the small size and thickness of the mandibular accessory foramina and nerve branches, as well as due to the dissection technique performed. The goal of this study was to identify and trace the CP branches from fresh human cadaver tissue samples using the Sihler's technique. Two fresh human cadaver samples were used. Samples were fixed in neutralized formalin, macerated in potassium hydroxide, decalcified in acetic acid, stained in Ehrlich's hematoxylin, destained in acetic acid, and cleared in glycerin. Both specimens skin was dissected. The Sihler's technique delineated all nerves three dimensionally and helped to disclose structures of small size and thickness. The TCN from the CP, stained in blue, innervated the posterior mandible in one of the two samples. These results confirmed that the CP may supply accessory innervation to the inferior border of the posterior mandible through the TCN. These findings illustrate variations of anatomy that may account for IANB failures in posterior mandibular teeth and allows for clinical decisions for implementing supplemental anesthetic techniques.

Different effects on bone strength and cell differentiation in pre pubertal caloric restriction versus hypothalamic suppression.

Hypothalamic amenorrhea and energy restriction during puberty affect peak bone mass accrual. One hypothesis suggests energy restriction alters hypothalamic function resulting in suppressed estradiol levels leading to bone loss. However, both positive and negative results have been reported regarding energy restriction and bone strength. Therefore, the purpose of this study was to investigate energy restriction and hypothalamic suppression during pubertal onset on bone mechanical strength and the osteogenic capacity of bone marrow-derived cells in two models: female rats treated with gonadotropin releasing hormone antagonists (GnRH-a) or 30% energy restriction. At 23 days of age, female Sprague Dawley rats were assigned to three groups: control group (C, n=10), GnRH-a group (n=10), and Energy Restriction (ER, n=12) group. GnRH-a animals received daily injections for 27 days. The animals in the ER group received 70% of the control animals' intake. After sacrifice (50 days of age), body weight, uterine and muscle weights were measured. Bone marrow-derived stromal cells were cultured and assayed for proliferation and differentiation into osteoblasts. Outcome measures included bone strength, bone histomorphometry and architecture, serum IGF-1 and osteocalcin. GnRH-a suppressed uterine weight, decreased osteoblast proliferation, bone strength, trabecular bone volume and architecture compared to control. Elevated serum IGF-1 and osteocalcin levels and body weight were found. The ER model had an increase in osteoblast proliferation compared to the GnRH-a group, similar bone strength relative to body weight and increased trabecular bone volume in the lumbar spine compared to control. The ER animals were smaller but had developed bone strength sufficient for their size. In contrast, suppressed estradiol via hypothalamic suppression resulted in bone strength deficits and trabecular bone volume loss. In summary, our results support the hypothesis that during periods of nutritional stress the increased vertebral bone volume may be an adaptive mechanism to store mineral which differs from suppressed estradiol resulting from hypothalamic suppression.

Attraction of specific thalamic input by cerebral grafts depends on the molecular identity of the implant.

The cerebral cortex of mammals differentiates into functionally distinct areas that exhibit unique cytoarchitecture, connectivity, and molecular characteristics. Molecular specification of cells fated for limbic cortical areas, based on the expression of the limbic system-associated membrane protein (LAMP), occurs during an early period of brain development. The correlation between this early molecular commitment and formation of specific thalamocortical connections was tested by using a transplantation paradigm. We manipulated the phenotype of donor limbic and sensorimotor neurons by placing them in different cortical areas of host animals. Labeling of transplanted tissue with the lipophilic dye 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine was used to assay host thalamic neurons projecting to the donor tissue. We found that limbic thalamic axons successfully projected into cortical transplants (i) when LAMP was expressed by early committed limbic cortical neurons, irrespective of their host location, and (ii) when LAMP was expressed by uncommitted sensorimotor progenitor cells whose fate was altered by their new host locale. Thus, the response of cortical neurons to both intrinsic and environmental cues that influence their molecular phenotype has an important anatomical correlate, the development of specific patterns of thalamocortical connectivity.

Induction of stress (heat shock) protein 70 and its mRNA in rat corneal epithelium by hyperthermia.

Because stress proteins are believed to play an important role in cellular repair and survival mechanisms, we investigated accumulation of the 70-kilodalton stress protein (SP70) and its mRNA in the rat corneal epithelium after hyperthermia. In the corneal epithelium of control rats, in situ hydridization with a radioactive probe for SP70 mRNA followed by autoradiography revealed very few silver grains. Eighteen hours after the rats were subjected to hyperthermia, the density of silver grains was greatly increased and this elevated level of corneal epithelium SP70 expression continued through 50 hours after heat treatment. Immunostaining for SP70 in the corneal epithelium was consistent with the in situ hybridization, being weak and mainly confined to the basal cells in control rats and increasing by 18 hours in the heat-treated rats. At 50 hours post-heat treatment, the immunostaining was denser than control in all corneal epithelial cells, especially in the apical portions of the basal and wing cells. These results suggest that SP70 may be an important factor in the response of the corneal epithelium to adverse environmental changes.