The Study Of Preparation And The Biocompatible Of Acellular Spinal Cord Scaffold

Objective This study was designed to maintain the integrity of the extracellular matrix and to keep its natural three-dimensional structure maximally in the context of complete removal of cells using the decellularization method to prepare excellent acellular spinal cord scaffolds, and to observe the growth of cells on the scaffolds after the prepared acellular spinal cord scaffolds were co-cultured with rat spinal cord neurons.Method 1. Ten SPF grade female Wistar rats were used to prepare acellular spinal cord scaffolds by chemical extraction method. Acellular spinal cord surface structure was observed by HE staining, myelin staining, and scanning electron microscope(SEM). DNA of the rat spinal cord scaffold tissue was extracted and DNA content was analyzed to make sure the successful decellularization of rat spinal cord scaffolds.2. Two experimental young SD rats were anaesthetized and the spinal cord tissues were removed, cut, and cultured in culture flasks with cell culture medium. Cultured cells were isolated and purified by enzyme digestion. Cells were identified by NSE immunohistochemical staining after being proliferated and passaged in vitro, and the growth status was observed and the growth curve was plotted. After 2 passages, the neuron cells were collected and co-cultured with acellular spinal cord scaffolds for 24 hours. MTT was used to observe the cell survival.3. The acellular spinal cord scaffolds were disinfected and trimmed into 0.5cm fragments. The third passage of rat spinal cord neurons were adjusted to a cell density of 1X105/ml and seeded on the acellular spinal cord scaffolds and cultured in DMEM medium supplanted with 10% FBS. The culture dishes were cultured in a 37℃, 5% CO2 incubator and cell growth was observed with an inverted microscope. The attachment and growth of neurons with the acellular spinal cord scaffolds was observed by HE staining. NSE and NEUN staining were used to determine whether the con-cultured cells were neurons and the 3D structure of the whole space was observed by SEM.Results 1. Rat acellular spinal cord scaffolds were prepared by chemical extraction. Acellular spinal cord scaffolds of Wistar rats were milky white, translucent, and flat cylindrical, with slightly shortened diameter and length, soft texture, increased toughness, and no peculiar smell. HE staining showed that mesh structure with unequal sizes was present on cross sections of the acellular spinal cord scaffolds and cellular components were basically removed, with no nuclear blue particles and red cytoplasm observed. Axons and myelin sheaths were removed and mesh walls were constituted of extracellular matrix with homogeneous pink staining and no residual cells inside and outside the mesh structure were seen. Myelin staining showed that no myelin constituents were shown on the rat acellular spinal cord scaffolds and some irregular blue myelin debris was present. Myelin SEM revealed the irregular mesh structure of the rat acellular spinal cord scaffolds and the layered arrangement of extracellular matrix, which formed irregular circular cavities. These excellent three-dimensional mesh-like structure showed thin walls, larger surface area, and interconnected porous structures. No cellular components were left, cells were completely removed, and axon and myelin sheath structures were totally disappeared. A certain roughness still existed on the extracellular matrix surface of the rat acellular spinal cord scaffolds. The nucleus was substantially removed with no purple-red nucleus existed, and the cellular components were completely removed. No DNAs were extracted from rat acellular spinal cord scaffold tissues, and no DNA bands were detected by DNA agarose gel electrophoresis, showing that the scaffolds were decellularized completely with no deoxyribonucleic acid substances and cells detected.2. NSE antibody staining of the prepared rat spinal cord neurons demonstrated that the cell body was brown and the nucleus was blue-purple and the cell shape was irregular with unequal length of neuritis, long axons, and fewer branches. These cells showed typical morphology of neurons, i.e., the prepared cells were indeed the rat spinal cord neurons.3. Rat spinal cord neurons were co-cultured with rat acellular spinal cord scaffolds and the HE staining results showed that the spinal cord neurons adhered to the acellular spinal cord scaffolds and the growth of the cells was good. Rat spinal cord neurons adhered to the pore structures of the WISTA rat acellular spinal cord scaffolds formed after the cells fell off. Cells fully grew in the 3-D channels to form aggregated cell regions, which did not affect the spatial structure of the rat acellular spinal cord scaffolds. Immunohistochemical staining of the co-culture showed that cell bodies of rat neurons stained were brown and the nuclei were blue-purple. Rat neurons attached to the pores of the acellular spinal cord scaffolds and grew. Cells were arranged closely, and the channel structures formed in acellular spinal cord scaffolds was fully filled by neurons. The cells accumulated in the channel area and the cells grew well, with a good growth state of the axons and an intact morphological structure, indicating the good biocompatibility and adaptability between the rat acellular spinal cord scaffolds and the rat spinal cord neurons.Conclusion 1. The integrity of the extracellular matrix and the natural three-dimensional structure of rat acellular spinal cord scaffolds were maximally maintained in the premise of complete decellularization by chemical extraction. Rat spinal cord neurons and rat acellular spinal cord scaffolds were co-cultured and the spinal cord neurons grew well and aggregated in the spatial channels of the acellular spinal cord scaffolds, with a good growth state of the axons and an intact appearance and structure.2. The structure of the acellular spinal cord scaffolds remained intact and rat neurons were closely arranged in the scaffolds. There was a good biocompatibility between the rat acellular spinal cord scaffolds and the rat spinal cord neurons.