Three-dimensional culture of bovine nucleus pulposus cells in protein matrices

Abstract

Introduction and objectives Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount of water, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). If a three-dimensional NPC-matrix platform mimicking the native NP is developed, it can be used in future research in tissue engineering and serve as a model platform to study NPCs in DDD. Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). The bNPCs were encapsulated in microspheres made of collagen type I and then cultured and fixed on days 3, 7, 10 and 14. Immunohistochemistry of collagen type II and histology including Alcian blue and hematoxylin and eosin (H&E) staining were done. Immunohistochemistry of NPC markers will also be done. The viability of the bNPCs in the microspheres was evaluated by live/dead staining. Besides the microsphere system, another three-dimensional culture system based on collagen-GAG co-precipitate will be investigated. In brief, chondroitin-6-sulfate, aminated type I collagen and bNPCs will be mixed together followed by centrifugation to collect the constructs, which will be cultured and evaluated in the same ways as the microspheres.

Results H&E showed that in the microspheres, the bNPCs were sparse in day 3 and became dense starting from day 7. At the periphery of the microspheres, a ring with very dense cells was formed since day 7 and grew thicker in later time points. The cell density in the ring was higher than that in the central region. Any difference between the cells in the two regions is yet to be investigated. Most of the bNPCs became highly elongated since day 10. Alcian blue staining was very weak and weak on days 3 and 7, respectively. It became moderate on days 10 and 14. These indicated that GAGs were slowly accumulated in the microspheres. Very intense staining for collagen type II was observed on days 7, 10 and 14. Live/dead staining showed that most of the cells were alive in all time points.

Conclusions The collagen microspheres supported the survival and proliferation of bNPCs and maintained some of the phenotypic characteristics of bNPCs, including the production of collagen type II and GAGs.