[Effect of tissue engineered bone implantation with vascular bundle and sensory nerve bundle on expression of neurokinin 1 receptor and vasoactive intestinal peptide type 1 receptor in vivo].

OBJECTIVE: Vascular bundle and sensory nerve bundle implantation can promote the osteogenesis of tissue engineered bone. To investigate whether vascular bundle and sensory nerve bundle implantation will affect the expressions of neurokinin 1 receptor (NK1R) and vasoactive intestinal peptide type 1 receptor (VIPR1). METHODS: Fifty-four 5-month-old New Zealand rabbits were selected. Autologous bone marrow was aspirated from the posterior iliac spine of rabbits, and the bone marrow mesenchymal stem cells (BMSCs) were proliferated in vitro. At the 3rd passage, the BMSCs were cultured in the osteogenic culture medium for 7 days. The tissue engineered bone was prepared by the combined culture of these osteoblastic induced BMSCs and beta tricalcium phosphate scaffold material. A 1.5 cm segmental bone defect was created at the right femur of rabbits. After the plate fixation, defects were repaired with sensory nerve bundle plus tissue engineered bone (group A, n = 18), with vascular bundle plus tissue engineered bone (group B, n = 18), and tissue engineered bone only (group C, n = 18). X-ray examination was used to evaluate the degree of the ossification. The expression levels of NK1R and VIPR1 were measured by the immunohistochemistry analysis and the mRNA expression of NK1R and VIPR1 by real-time PCR at 4, 8, and 12 weeks after operation. RESULTS: The better osteogenesis could be observed in group A and group B than in group C at all time points. X-ray scores were significantly higher in group B than in groups A and C (P < 0.05) at 4 weeks, and in groups A and B than in group C (P < 0.05) at 8 and 12 weeks. The mRNA expressions of NK1R and VIPR1 were highest at 8 weeks in groups A and B and gradually decreased at 12 weeks (P < 0.05); the expressions were higher in groups A and B than that in group C (P < 0.05), and in group B than group A (P < 0.05). Immunohistochemistry analysis showed that the expressions of NK1R and VIPR1 were highest at 8 weeks in 3 groups, and the expressions were higher in groups A and B than in group C. CONCLUSION: Implanting vascular bundles into the tissue engineered bone can significantly improve the expression levels of NK1R and VIPR1. It is an ideal method to reconstruct composite tissue engineered bone.

[Experimental study on construction of neurotization tissue engineered bone for repairing large bone defects in rabbit].

OBJECTIVE: Construction of viable tissue engineered bone is one of the most important research fields in the clinical application of bone tissue engineering, to investigate the function of nerve factors in bone tissue engineering by cell detection in vitro and construction of neurotization tissue engineered bone in vivo. METHODS: Fifty-four healthy New Zealand white rabbits, male or female, weighing 2-3 kg, were involved in this study. Bone marrow mesenchymal stem cells (BMSCs) from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates, using the LG-DMEM complete medium as control. The proliferation and osteogenic differentiation of the cells were observed and tested by the MTT assay, alkaline phosphatase (ALP) stain, and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in beta tricalcium phosphate (beta-TCP) biomaterial scaffold and cultured for 72 hours, then the beta-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups (n = 18): sensory nerve bundle (group A) or motor nerve bundle (group B) were transplanted into the side groove of beta-TCP scaffold, group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th, 8th, and 12th weeks after operation. Bone mineral density (BMD) detection and S-100, calcitonin gene-related peptide (CGRP) immunohistochemistry stain were used at the 12th week to evaluate the effects of bone formation and discuss the mechanism. RESULTS: MTT assay indicated that the absorbance (A) value of each group increased with culture time. From the 6th day, the A values of both the sensory nerve and motor nerve homogenate groups were lower than that of the control group, showing significant difference (P < 0.01). On the 8th and 10th days, the A value of the sensory nerve homogenate group was lower than that of the motor nerve homogenate group, showing significant difference (P < 0.05). ALP stain and collagen type I immunocytochemistry identification indicated that the positive cells in both the sensory nerve and motor nerve homogenate groups were less than that of control group after culturing 7 days. And the positive expression of collagen type I was just visible in the cells of control group. The Yang's scores increased gradually in three groups, the score of group A was significantly higher than those of group B and group C (P < 0.01) at the 8th week. The BMD value of group A was significantly higher than those of group B and group C (P < 0.01) at the 12th week. The S-100 and CGRP expressions were high in group A, and low in group B and group C. CONCLUSION: Homogenates of sensory nerve and motor nerve have inhibitory effects on the proliferation and osteogenic differentiation of BMSCs. The osteogenesis and remodeling of the neurotization tissue engineered bone are more closely related with sensory nerves.

Chemical and electric transmission in the carotid body chemoreceptor complex

Carotid body chemoreceptors are complex secondary receptors. There are chemical and electric connections between glomus cells (GC/GC) and between glomus cells and carotid nerve endings (GC/NE). Chemical secretion of glomus cells is accompanied by GC/GC uncoupling. Chemical GC/NE transmission is facilitated by concomitant electric coupling. Chronic hypoxia reduces GC/GC coupling but increases G/NE coupling. Therefore, carotid body chemoreceptors use chemical and electric transmission mechanisms to trigger and change the sensory discharge in the carotid nerve.

Survival of adult rat retinal ganglion cells with regrown axons in peripheral nerve grafts: a comparison of graft attachment with suture of fibrin glue.

OBJECT: The goal of this study was to examine whether the method of attachment of a peripheral nerve graft would have an effect on retinal ganglion cell (RGC) regeneration. METHODS: The number of adult rat RGCs with regrown axons in a peripheral nerve graft was compared under two grafting conditions: 1) attachment of the graft to the optic nerve stump made using a suture; and 2) attachment made using fibrin glue. Counts of RGCs retrogradely labeled with FluoroGold from the grafts 1 month after attachment revealed approximately seven times the number of RGCs in the fibrin-glue group compared with the suture group. CONCLUSIONS: The use of fibrin glue may be a useful tool for enhancing the regrowth of central nervous system neuron axons into peripheral nervous system grafts.

Afferents of cranial sensory ganglia pathfind to their target independent of the site of entry into the hindbrain.

In vertebrates, sensory neurons interconnect a variety of peripheral tissues and central targets, conveying sensory information from different types of sensory receptors to appropriate second-order neurons in the central nervous system (CNS). To explore the possibility that the different rhombomere environments where sensory neurons enter into the hindbrain affect the pathfinding capability of growth cones, we studied the development of the VIIIth ganglion afferent both in vivo and in vitro. We focused on the vestibular nerve because it is the only cranial nerve projecting to the cerebellum, allowing for ready identification from its pattern of projection. Embryonic rat brain was cut along the dorsal midline and, with the VIIIth and Vth ganglia still attached, flat mounted and visualized with antibodies specific for sensory ganglia. Axons reached the cerebellar primordium at embryonic day (E) 13, then splayed out towards the edges of the rhombic lip of rostral hindbrain. In vitro, the VIIIth ganglion showed development similar to that in vivo and innervated the cerebellum, an appropriate target, indicating that mechanisms for axon guidance and target recognition are preserved in vitro. When the VIIIth ganglion was transplanted to the position of the Vth ganglion, axons from the transplanted ganglion entered the cerebellar primordium with a trajectory characteristic of the VIIIth nerve. These results indicate that the central projection pattern of the VIIIth nerve is not affected by the environment of nerve entry into the brainstem, suggesting that axons of sensory cranial ganglion intrinsically possess the capacity to find their target correctly.