PREPARATION OF BIONIC COLLAGEN-HEPARIN SULFATE SPINAL CORD SCAFFOLD WITH THREE-DIMENSIONAL PRINT TECHNOLOGY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGRenkun , TUYue , ZHAOMingliang , CHENChong , LIANGHaiqian , WANGJingjing , ZHANGSai ,  LIXiaohong

Institute of Traumatic Brain Injury and Neuroscience, Neurotrauma Repair Key Laboratory of Tianjin, the Affiliated Hospital of Logistics University of Chinese People's Armed Police Forces, Tianjin, 300162, P.R.China;

Corresponding author：

LIXiaohong, Email: lixiaohong12@hotmail.com

Keywords：

Bionic spinal scaffold; Three-dimensional print; Neural stem cells; Biological compatibility

DOI：

10.7507/1002-1892.20150218

Video：

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Objective To prepare bionic spinal cord scaffold of collagen-heparin sulfate by three-dimensional (3-D) printing, and provide a cell carrier for tissue engineering in the treatment of spinal cord injury. Methods Collagen-heparin sulfate hydrogel was prepared firstly, and 3-D printer was used to make bionic spinal cord scaffold. The structure was observed to measure its porosity. The scaffold was immersed in simulated body fluid to observe the quality change. The neural stem cells (NSCs) were isolated from fetal rat brain cortex of 14 days pregnant Sprague-Dawley rats and cultured. The experiment was divided into 2 groups: in group A, the scaffold was co-cultured with rat NSCs for 7 days to observe cell adhesion and morphological changes;in group B, the NSCs were cultured in 24 wells culture plate precoating with poly lysine. MTT assay was used to detect the cell viability, and immunofluorescence staining was used to identify the differentiation of NSCs. Results Bionic spinal cord scaffold was fabricated by 3-D printer successfully. Scanning electron microscope (SEM) observation revealed the micro porous structure with parallel and longitudinal arrangements and with the porosity of 90.25%±2.15%. in vitro, the value of pH was not changed obviously. After 8 weeks, the scaffold was completely degraded, and it met the requirements of tissue engineering scaffolds. MTT results showed that there was no significant difference in absorbence (A) value between 2 groups at 1, 3, and 7 days after culture (P>0.05). There were a lot of NSCs with reticular nerve fiber under light microscope in 2 groups;the cells adhered to the scaffold, and axons growth and neurosphere formation were observed in group A under SEM at 7 days after culture. The immunofluorescence staining observation showed that NSCs could differentiated into neurons and glial cells in 2 groups;the differentiation rate was 29.60%±2.68% in group A and was 10.90%±2.13% in group B, showing significant difference (t=17.30, P=0.01). Conclusion The collagen-heparin sulfate scaffold by 3-D-printed has good biocompatibility and biological properties. It can promote the proliferation and differentiation of NSCs, and can used as a neural tissue engineered scaffold with great value of research and application.

Citation： ZHANGRenkun, TUYue, ZHAOMingliang, CHENChong, LIANGHaiqian, WANGJingjing, ZHANGSai, LIXiaohong. PREPARATION OF BIONIC COLLAGEN-HEPARIN SULFATE SPINAL CORD SCAFFOLD WITH THREE-DIMENSIONAL PRINT TECHNOLOGY. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(8): 1022-1026. doi: 10.7507/1002-1892.20150218 Copy

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1. Freund P,Curt A,Friston K,et al.Tracking changes following spinal cord injury:insights from neuroimaging.Neuroscientist,2013,19(2):116-128.
2. Shrestha B,Coykendall K,Li Y,et al.Repair of injured spinal cord using biomaterial scaffolds and stem cells.Stem Cell Res Ther,2014,5(4):91.
3. Ma W,Fitzgerald W,Liu QY,et al.CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.Exp Neurol,2004,190(2):276-288.
4. Lavik E,Teng YD,Snyder E,et al.Seeding neural stem cells on scaffolds of PGA,PLA,and their copolymers.Methods Mol Biol,2002,198:89-97.
5. Möllers S,Heschel I,Damink LH,et al.Cytocompatibility of a novel,longitudinally microstructured collagen scaffold intended for nerve tissue repair.Tissue Eng Part A,2009,15(3):461-472.
6. Madigan NN,McMahon S,O'Brien T,et al.Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds.Respir Physiol Neurobiol,2009,169(2):183-199.
7. 杨志明.加快发展3-D打印技术、扩展修复重建外科应用领域.中国修复重建外科杂志,2014,28(3):265.
8. Michalski MH,Ross JS.The shape of things to come:3D printing in medicine.JAMA,2014,312(21):2213-2214.
9. Ishikawa T,Suzuki H,Ishikawa K,et al.Spinal cord ischemia/injury.Curr Pharm Des,2014,20(36):5738-5743.
10. Krishna V,Konakondla S,Nicholas J,et al.Biomaterial-based interventions for neuronal regeneration and functional recovery in rodent model of spinal cord injury:a systematic review.J Spinal Cord Med,2013,36(3):174-190.
11. Deng LX,Walker C,Xu XM.Schwann cell transplantation and descending propriospinal regeneration after spinal cord injury.Brain Res,2014.[Epub ahead of print].
12. Gao M,Lu P,Bednark B,et al.Templated agarose scaffolds for the support of motor axon regeneration into sites of complete spinal cord transection.Biomaterials,2013,34(5):1529-1536.
13. Mothe AJ,Tam RY,Zahir T,et al.Repair of the injured spinal cord by transplantation of neural stem cells in a hyaluronan-based hydrogel.Biomaterials,2013,34(15):3775-3783.
14. Murray AJ.Axon regeneration:what needs to be overcome? Methods Mol Biol,2014,1162:3-14.
15. Fansa H,Keilhoff G.Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects.Neurol Res,2004,26(2):167-173.
16. Zhao S,Wang Z,Chen J,et al.Preparation of heparan sulfate-like polysaccharide and application in stem cell chondrogenic differentiation.Carbohydr Res,2015,401:32-38.
17. Yuying B,Patra P,Faezipour M.Structure of collagen-glycosaminoglycan matrix and the influence to its integrity and stability.Conf Proc IEEE Eng Med Biol Soc,2014,2014:3949-3952.
18. Nikitovic D,Mytilinaiou M,Berdiaki A,et al.Heparan sulfate proteoglycans and heparin regulate melanoma cell functions.Biochim Biophys Acta,2014,1840(8):2471-2481.
19. Tam RY,Fuehrmann T,Mitrousis N,et al.Regenerative therapies for central nervous system diseases:a biomaterials approach.Neuropsychopharmacology,2014,39(1):169-188.
20. Zamani F,Amani-Tehran M,Latifi M,et al.Promotion of spinal cord axon regeneration by 3D nanofibrous core-sheath scaffolds.J Biomed Mater Res A,2014,102(2):506-513.
21. Ribeiro-Samy S,Silva NA,Correlo VM,et al.Development and characterization of a PHB-HV-based 3D scaffold for a tissue engineering and cell-therapy combinatorial approach for spinal cord injury regeneration.Macromol Biosci,2013,13(11):1576-1592.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION OF BIONIC COLLAGEN-HEPARIN SULFATE SPINAL CORD SCAFFOLD WITH THREE-DIMENSIONAL PRINT TECHNOLOGY

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