Experimental Study On Demineralized And Decellular Bone As Scaffold For Intervertebral Disc Anulus Fibrosus Tissue Engineering

Low back pain is one of the common diseases and a frequently occurring illness in modern society. Importantly, not only in the elder population, in the younger population the prevalence of low back pain is extremely high and is one of the major causes of disability . Intervertebral disc disorder is a major contributor to low back pain. Of the intervertebral disc disorders, disc degeneration disease(DDD) is one of the major causes of low back pain, such as disc herniation. As the aging process of the population in our country , DDD will impact the people in working and living more and more severely.Current conventional treatment of DDD includes medication, steroid injection, physical therapy, and surgery which is the most effective and common method. Recent progress in minimally invasive operations for disc herniation, such as endoscopic discectomy and percutaneous discectomy, still damage the disc structure. This, in turn, may cause further degeneration that eventually leads to instability of the motion segment that requires interbody fusion to stabilize those segments. After interbody fusion,the spinal segments lost the motor function which is against to the functional reconstruction purpose of modern medicine. The long-term results of the intervertebral disc prostheses are not satisfactive. The intervertebral disc allografting has the problems of immunological rejection and pathophoresis. More Importantly, it is short of donators. Theoretically, treatments that strategize to repair or regenerate the intervertebral disc biologically appear promising as a future treatment option. Therefore, to study and develop tissue engineered intervertebral disc for biologically repairing of intervertebral disc is an ideal therapeutic regimen.The intervertebral disc is composed of a tough outer ring(anulus fibrosus) and a gelatinous inner core(nucleus pulposus). Theoretically, the investigation of the treatment of anular lesions is a logical initial step because no method of restoration of the nucleus pulposus is likely to be successful in the setting of a compromised anulus fibrosus. Once anulus fibrosus repair is achievable, a logical approach to the treatment of the spectrum of disc disease will become possible. In an advanced disease stage, with an isolated anular tear without frank herniation, repair of the anulus fibrosus using cell-based anulus fibrosus tissue, followed by an nucleus pulposus-stimulating treatment such as an injection of growth factor, may be appropriate. For severe disc disease, in the presence of frank herniation of the nucleus pulposus, combining repair of the anulus fibrosus and nucleus pulposus with cellbased anulus fibrosus and nucleus pulposus tissue may be necessary.Demineralized bone matrix gelatin(DBMG) has been used as scaffold in the study of cartilaginous tissue engineering successfully. The researchers have got cartilaginous tissues which confirms that DBMG is good at easily to gain, no toxical material produced in degradation process, and to maintain biological and biomechanical nature after freezing for a long time. In addition, decellular natural tissues have been extensively and successfully used as scaffold in the study of tendon and blood vessel tissue engineering.Is it possible that demineralized and decellular bones can be used as scaffold in the research of intervertebral disc anulus fibrosus tissue engineering? How about the characteristics and efficiency of them? If the seed cells have been planted into them can they adhere, proliferate and functionate? The studies in this field have not been reported in the references before.To answer the questions above, we emploied the techniques of tissue engineering, molecular biology, cell culture and et al to develop the serial studies in this field which begins with manufacture of the demineralized and decellular bones. Firstly, the scaffolds of anulus fibrosus were manufactured, which have the appropriate physical and chemical character and low immunogenicity. Secondly, the rabbit intervertebral disc anulus fibrosus cells were isolated and cultured. In vitro, they were cultured in monolayer and amplificated. Seed cells with stable phenotype, better proliferative activity and sufficient quantity were provided. Thirdly, the seed cells were planted into the scaffold with two difference methods, static precipitation method and fibrin gelatum noculation method. In this study we confirmed that the cells could adhere and proliferate in this scaffold with both methods. Especially in the second one, it seems that the cells could adhere more in quantity, distribute more uniform and proliferate more quickly. Eventually, the cell-scaffold composites were cultured in vitro and in vivo of athymic mouse subcutaneouly. The cultured productions were identified as anulus fibrosus like tissues, especially thoese in athymic mouse, which implied further that the seed cells could proliferate and posses biological function in the scaffold.The main conclusions are summaried as follow:1. The anulus fibrosus scaffolds with porous structure and anulus fibrosus outline form were obtained after the rabbit bones were demineralized, decellular and reshaped. The outer layer of the scaffolds was compact with mechanics intensity and the inner layer was porous and convenient for implanting of the seed cells. The internal spacing provides the space of the nucleus pulposus tissue engineering. The average pore size, porosity and suction force of the materials achieved the requirement of cell scaffolds in tissue engineering researchs. The anulus fibrosus scaffolds had some degree of flexibility, degradation property and low immunological rejection response by the recipient. So, these scaffolds can be used in the anulus fibrosus tissue engineering studies performed below.2.After the anulus fibrosus tissues were disconnected from the rabbit intervertebral disc, the enzyme digestion with two steps including trypsinase and type II collagenase was emploied to isolate the anulus fibrosus cells. Then, the cells were cultured in monolayer. In the primary culture, the cells proliferated slowly. In the first generation, they were similar to the primary cultured cells in morphous, vital force and phaenotype. In addition, the first generation cells proliferated faster than the primary cultured cells, which indicated that we could harvest enough cells for anulus fibrosus tissue engineering studies quickly. Through this experiment, the seed cells with enough quantity and stable phenotype could be provided for the subsequent researchs.3.The seed cells coming from the first generation anulus fibrosus cells were planted into the scaffolds manufactured by demineralized and decellular bones in two inoculation ways which were static precipitation method and fibrin gelatum noculation method. In both methods, the cells planted into the scaffolds could adhere and proliferate. But it seems that the cells could adhere more in quantity, distribute more uniform and proliferate more quickly in the cell-scaffold composites manufacture by fibrin gelatum noculation method. So, the further culture would be benefited from this experimental result.4. The cell-scaffold composites manufactured by fibrin gelatum noculation method were cultured in vitro. The productions were analysed and proved that the cells in them could retain the phaenotypes, proliferate continuosly and perform biological functions, so they were identified as anulus fibrosus like tissues. Because the cells did not proliferate after 2 months and the scaffolds were not absorpted obviously, a better culture system should be emploied.5. The cell-scaffold composites manufactured by fibrin gelatum noculation method were cultured in vivo of athymic mouse subcutaneouly. The productions were analysed and proved that the cells in them could retain the phaenotypes, proliferate continuosly and perform biological functions. With most of the scaffolds absorpted, The productions were more like the anulus fibrosus tissues than those cultured in vitro. Because the cells could proliferate longer, produce more functional productions, this in vivo culture system was better than the in vitro one, which provided a better choice for biological repair of anulus fibrosus.