Research On The Effects Of TGF-β₃and Dental Pulp Stem Cells In Repair Of Rabbit Facial Nerve Injury

ObjectiveFacial nerve injury is common clinically, and the function can not be recoveredperfectly after facial nerve injury, which severely affected psychosomatic health andquality of life of patients and families. With the rapid development of the stem cellresearch, the reporter in the literature was more about using bone marrow stem cells forthe treatment of spinal cord and brain injury, but the method that to treatment peripheralnerve injuries using stem cells has not been reported. The purpose of this study was toinvestigate the effects of TGF-β₃and dental pulp stem cells in the regeneration of rabbitfacial nerve, to explore possible mechanism of action, and to provide a new method forthe treatment of facial nerve injury.Methods:（1） Dental pulp stem cells were separated and cultured in vitro from anterior teethand molar pulp tissue of New Zealand rabbit, and to test the cells clone forming rate, tomeasure cells growth curve by counting the number of cells, did HE staining andimmunhistochemistry staining of Vimentin, osteonectin、dsp and CD44. To identificationproliferation capacity and multiple differentiation potential of biological characteristics ofDPSCs.（2）12New Zealand adult rabbits were selected randomly. These rabbits weredivided into two groups （the normal groups and the model groups）.7days,14days afteroperation, a series of examinations were performed to evaluate the model, includinghistopathology testing, animal behavior observation, neuroelectrophysiological methods.（3）48New Zealand adult rabbits（a total of96facial nerves）were randomly dividedinto4groups，respectively in treatment group （silicone guidance channel with collagenprolein sponge was filled with TGF-β₃+DPSCs）, control group1（silicone guidancechannel with collagen prolein sponge was filled with TGF-β₃），PBS control group2 （silicone guidance channel with collagen protein sponge was filled with PBS） and thenormal group．A7mm nerve defect was set up in the buccal branch of facial nerve andwas bridged with three sorts of artificial nerves. One week, one month, three months afteroperation， a series of examinations were performed respectively, including grossmorphology, histopathology testing, neuroelectrophysiological examination, GFAP andS100immunohistochemical stain.Results:Part one:1) Primary cultured rabbit dental pulp cells were fibroblast-like cells, afusiform or polygonal. characteristics were no difference between subcultured cell andprimary culture under a light microscope. The morphology character of the cells insubcultllring was as same as that in primary culture. Cell survival rate in the cell numbersfrom the first generation to the third generation were:94.7%,95.8%,95.2%. DPSCscould form clone after low density seeding.The cells clone forming rate was10-21/103cells.2) DPSCs have a perfect growing ability after multiple subculture.24holeplate counting method that rabbit dental pulp cells incubation period was short, thenentered the logarithmic growth phase, the third generation cell in five days to reach thelogarithmic growth phase, in about eighth days the cells reach the platform period. Theresult of the cell cycle: G1DNA content of80.4%G2; the content of DNA15.3%; S DNAcontent accounted for:4.3%.3) Immunhistochemistry staining showed that Vimentin、CD44、osteonectin and dsp of dental stem cells were positive in Clone of the original andpassaged.Part two:1) All experiments were accomplished successfully, operation process wassmooth, animal survival rate was100%, and the success rate of modeling was100%.2)After modeling, all experimental animals appeared facial nerve paralysis symptoms in theoperation side. At2weeks after operation, the face on the operative side become mildatrophy. Facial whiskers motor function score showed that the scores of operative sidewere0.25±0.05, while the normal side scores were4points, there was a significantdifference （t=-249.16, P＜0.05）.3) The neuroelectrophysiological examinations revealedthat the latency of nerve and muscle action conduction in the normal control groups were（1.47±0.42） ms, the wave amplitude of nerve and muscle action conduction in thenormal control groups were （11.32+5.36） mV. At one week、two weeks after operation,lateral nerve action potentials were unable to lead.4) Facial nerve HE staining showednormal facial nerve fibers elongated strip, arranged in neat rows, the myelin-intensive, no degeneration, the epineurium continuous; While surgery group facial nerve fiberdiscontinuity, ends part of the demyelination and the outer membrane was loose, the nervefiber was swollen, plate layer structure was loose, axonal vacuolization.5) Electronmicroscopy results showed that the normal facial nerve axons arranged in neat rows,cross-section showing circular, structural integrity of the myelin sheath, uniform thickness,dense lamellar arrangement, clear nucleus, clear Ranvier’s node; At one week afteroperation, model group showed that the facial nerve sheath dissociation, platy structureblurred to disappear resulting in the formation of oval-shaped body, appeared nerve fiberballs，low electron density in the axoplasm; At two weeks after operation, the modelgroup results showed that the axon swelling obviously, the number of microtubules andactin filaments significantly reduced, mitochondria were vacuolated, lower electrondensity in myelin sheath， disordered arrangement of nerve fibers, epineuriumdiscontinuity and lamellar detachment.Part three:1) At three months after operation, gross morphological observationfound that the diameter was almost the same between the regenerating nerve and near、distal nerve stem in the experimental group， no neuroma formation, adventitialangiogenesis was rich, tough texture.2) At three months after operation, HE stainingshowed that in the experimental group, the facial nerve membrane integrity, nerve fibersarranged in neat rows, dense nuclei. But in the control group1, the nerve fibers atrophy,regeneration fewer nerve fibers and distort obviously，was convoluted; in the controlgroup2, some axons disappeared, the nerve fibers arranged in disorder, the myelin sheathof uneven thickness.3) Electron microscopy results showed that the regenerated fibers inthe experimental group were mainly myelinated never fiber. The layer structure of myelinsheath was clear, and there were rich organells axoplasma. But in the control group1，themyelin dysplasia，disorder of the lamellar structure，myelinated nerve fibers with irregularshape, the degeneration of nerve fibers.4) Image analysis results showed that the totalnumber of regeneration of nerve fibers in the experimental group were more than theother control groups, statistical analysis was significant （P＜0.05）. Diameter ofregenerating nerve fibers in the experimental group were greater than the other controlgroup, statistical analysis was significant （P＜0.05）.5) At three months after operation,the neuroelectrophysiological examinations revealed that the latency of nerve and muscleaction conduction in the treatment group was shorter than the other control groups[thetreatment group:（1.96±0.32） ms, the control group1（2.35±0.41） ms, the control group2（3.42±0.55） ms], but the wave amplitude of nerve and muscle action conduction in the treatment group was obviously higher than that in the control group[the treatment group:（11.06±3.25） mv, the control group1（8.40±1.68） mv, the control group2（4.62±0.77）mv, P＜0.05].6) Facial nerve nuclear all appeared GFAP positive cells in each group,and the number of GFAP positive cells：TGF-of β₃+DPSCs experimental group>TGF-of β₃control group1> PBS control group2（P＜0.05）; Facial nerves all appearedS100positive cells in each group，and the number of S100positive cells：TGF-β₃+DPSCs experimental group> TGF-β₃the control group1> PBS control group2（P<0.05）.Conclusion:（1） DPSCs cultured in our experiment have both strong self-proliferation potentialand stable vital activity. Thus these cells are considered to be suitable for long-termculture in a large amount.（2） The cell clone rate of DPSCs in the first、second and third generation is highest.These clone cells can be used as the source of grafting.（3） Animal model established in this study is simple, stable, good repeatability,postoperative animal survival rate and modeling success rate is high. And these evaluationmethods are systematic, reliable.（4） DPSCs can be applied as seed cells for peripheral nerve tissue engineering.（5） The comination of TGF-β₃and DPSCs can improve the effects on the repair offacial nerve injury.（6） The artificial nerve made of chitosan guidance channel, collagen protein sponge,DPSCs and nerve growth factor is the most effective in repairing nerve defect and for itaccorded with the histological structure of nerve. Therefore it may provide a new methodfor repairing the pepripheral nerve defect in clinic.