Experiment Study On Construction And Correlated Properties Of Acellular Scaffold Of Spinal Cord

Backgrounds :Spinal cord injury usually results in devastating and permanent loss of function below the injured place. Because the CNS axons lack the ability for spontaneous regeneration--further compounded by chemical (myelin inhibitors) and physical (e.g. glial scar) barriers to regeneration after spinal cord injury, patients normally experience poor functional recovery.Several methods have been investigated to overcome this hostile environment for regeneration and to promote partial functional recovery. Strategies to promote axonal extension through a site of injury include both the provision of nervous system growth factors and the implantation of substrates to support axon extension, such as cellular grafts. In general, however, the growth of axons is highly random and does not extend beyond the lesion site and into host tissue.Recently researchers have realized that it is difficult to achieve complete functional recovery relying on a single method. This has presented tissue engineering technology as an alternative strategy for the treatment of spinal cord injury. The scaffold can guide the linear growth of axons across a site of injury, in addition to providing neurotrophic and/or cellular support. This will help retain the native organization of regenerating axons across the lesion site and into distal host tissue, eventually increasing the probability of achieving function recovery.Various natural and synthetic polymeric materials have been used to promote the functional recovery after spinal cord injury. Nevertheless, further improvements are needed for all previously-reported approaches, which mimic a native extracellular matrix. Acellular scaffolds are among the various materials that have been recently used in tissue reconstruction. Acellular scaffolds are the noncellular part of a tissue and consist of such proteins as collagen and carbohydrate structures secreted by resident cells. They can be transplanted without rejection and can provide a conductive environment for normal cellular attachment, migration, proliferation, differentiation and angiogenesis as well as a framework for tissue regeneration, since they are completely replaced by the host tissue. In the last few years, acellular matrices have been successfully used to substitute and repair skin, bladder, urethra, small bowel, cardiac valve, blood vessel, skeletal muscle,peripheral nervous defects, among others. In an attempt to mimic the regenerative capacity of the spinal cord graft, we have investigated acellular spinal cord that provides the physical pathway for axonal regeneration.Objective:The acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The program of decellulated was made and optimized. The acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis. The biological safety of acellular spinal cord scaffold was detected by component analysis, immunogenicity analysis, cytotoxicity, histocompatibility, blood compatibility, general toxicity reaction, thus provide rationale for constructing the ideal tissue engineering scaffold of spinal cord.Materials and Methods1. The program of acellular spinal cord scaffold preparation: the acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The effect of acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis,thus the optimized program was determined.2.Component analysis and immunogenicity detection: Specimens were embedded in tissue-freezing medium, with temperature fixed at -20℃. Sections of 8 mm thick were obtained and routinely stained with immunostained for collagen typesⅣ, Laminin(LN), Fibronectin(FN).A bilateral surgical approach was made to implant the acellular spinal cord scaffolds into the subcutaneous back skins of SD rat. Thereafter, a small skin incision was made (10 mm long), and a pocket was created through blunt dissection. The scaffolds were then implanted through these skin incisions subcutaneously into the mid-portion of the back areas. The incision was sewed using conventional cotton sutures. The tissue was obtained at 1,2,3 and 4w after the operation, with inflammatory reaction evaluated by HE stain. The immunogenicity of acelular scaffold was tested by immunohistochemical examining the intensity of CD4+ and CD8+ cells that infiltrated the allografts.3. Cytotoxicity and general toxicity reaction investigation:The cytotoxicity was tested through co-incubation of scaffolds with NIH 3T3 cells. The blood compatibility was detected by hemolysis test and clotting time.With the haemolysis rate <5%, the scaffold is qualified for becoming bio-tissue engineering materials. With the haemolysis rate≥5%,the scaffold has hemoclasis. The general toxicity was evaluated through pyrogen test and general toxic reaction.4. The acellular spinal cord scaffold combinated neural stem cells to construct tissue engineering spinal cord: the neural stem cells was seeded in the scaffold by different concentration gradient and then co-cultured in incubator. The cell survival status was observed by microscope,then calculated the adhesive rate and drawed growth curve of cells.Results and ConclusionsMacroscopic ObservationThere was a large amount of white-colored floss secreting from spinal cord during decellulation. After being treated, the spinal cord scaffold became ivory-white and translucent, and yet still shaped as a circular cylinder. However, the diameter shrank to 2/3-4/5 of that of the original spinal cord and the strength decreased slightly, although the tenacity remained unchanged and the viscosity increased slightly.HE StainingNormal spinal cord has generous neurons ,glial cells and myelin sheaths. In cross section, a network of the extracellular matrix was seen in the scaffold. The cells, myelin and axons disappeared after the spinal cord was treated with the detergents TritonX-100 and deoxycholate. Typical network of empty tubes were viewed in longitudinal sections .Myelin StainingNormal myelin sheath of spinal cord is black and has regulation shape. In acellular spinal cord, either no myelin sheaths is observed or only a small quantity of myelin-sheath pieces is detected.SEM AnalysisIn the scaffold, the cells have been removed completely, although the extracellular matrix and the pore have remained to form three diamensional network structures.The pore and the channel of scaffold diameter was 6-150μm and 119±26μm respectively.ImmunohistochemistryPositive reaction to LN,FN and IV collagen is seen in both normal spinal cord and acellular spinal cord.The staining is weaker in acellular scaffold than in normal spinal cord, which indicates that the majority of the extracellular matrix is preserved after the decellulation treatment of spinal cords.Biocompatibility in VitroA haemolysis rate <5% qualifies scaffold as bio-tissue engineering materials. After scaffolds were co-incubated with NIH 3T3 cells for 72 h, the NIH 3T3 cells showed no signs of cytotoxicity (loss of adherence, nuclear condensation, and cell soma contraction) and cells proliferated normally compared to cells in control wells, expanding from approximately 50–100% confluency within 72 h.Histocompatibility in VivoThe lymphocyte, neutrophilic granulocyte and fibroblast were seen in control groups and experimental group animals. The degree of infiltration in experimental groups was significantly weaker than in control groups after 1 week of implantation. There was no obvious increase of infiltrated cell of implantation and the neutrophilic granulocyte had vanished after 4 weeks. However, there was multiplicity lymphocyte and neutrophilic granulocyte infiltrated in control groups after 4w of implantation.There were sparing CD4+ and CD8+ leukomonocyte infiltration 1week and 2 weeks after implantation. Moreover, there were no obvious increase in experimental groups. There were massive CD4+ and CD8+ leukomonocyte infiltratration after implantation, and the staining intensity of positive cells were obviously stronger compared with the experimental groups.Our study first showed that segments from the spinal cord of Sprague–Dawley rats can be successfully extracted to become acellular. The outcome of the extraction was monitored by morphological methods and immunohistochemistry. The extraction procedure involved the removal of myelin following the weil's myelin staining, and cells, leaving largely intact ECM. Immunohistochemistry analysis revealed the presence of Laminin, Fibronectin and IV collagen in the ECM.The scaffold of spinal cord is an emulated three-dimensional natural spinal cord which has fundamentally distinct and innate superiority over biological degradation materials. It is easy to obtain and is not confined by length or caliber. It can be isoloci transplantation and anatomy transplantation.Acellular spinal cord scaffolds created in this study have a number of positive properties that can potentially support axonal regeneration after nervous system injury. The scaffolds are soft and flexible, containing linear guidance pores extending through their full length. Because the scaffolds are stable under physiological conditions, there is no risk of introducing toxic molecules to the site of injury. Based on current findings, we believe that extracted spinal cord could be used as allografts, with the possibility of becoming useful for spinal cord repair in the future. Ongoing work in vivo will test their ability to support axonal regeneration after spinal cord injury. The experiments of functional nerve recovery as well as microscopical (and morphometric) analysis will be evaluated in more detail.