SPINE Volume 31, Number 9, pp 959 –966 ©2006, Lippincott Williams & Wilkins, Inc. Platelet-Rich Plasma (PRP) Stimulates the Extracellular Matrix Metabolism of Porcine Nucleus Pulposus and Anulus Fibrosus Cells Cultured in Alginate Beads Koji Akeda, MD,* Howard S. An, MD,* Rajeswari Pichika, PhD,* Mohamed Attawia, MD,§ Eugene J.-M. A. Thonar, PhD,*†‡ Mary Ellen Lenz, MS,† Atsumasa Uchida, MD, PhD, and Koichi Masuda, MD*† Study Design. In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabo- lism of porcine intervertebral disc cells. Objectives. To determine whether platelet-rich plasma is effective in stimulating cell proliferation and extracellu- lar matrix metabolism by porcine disc cells cultured in alginate beads. Summary of Background Data. Platelet-rich plasma is used to accelerate wound healing and tissue regenera- tion. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and mor- phogenesis. Individual growth factors present in platelet- rich plasma have been demonstrated to affect the metab- olism of intervertebral disc cells. Methods. Platelet-poor and platelet-rich plasma was iso- lated from fresh porcine blood using a commercially avail- able platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads contain- ing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% plate- let-rich plasma. The synthesis of proteoglycans and colla- gen, the accumulation of proteoglycans, and the DNA con- tent were biochemically assessed. Results. Platelet-rich plasma had a mild stimulatory ef- fect on cell proliferation of intervertebral disc cells. Platelet- rich plasma treatment significantly upregulated proteogly- can and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma. Conclusions. Platelet-rich plasma was effective in stim- ulating cell proliferation and extracellular matrix metabo- lism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleus pulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addi- tion, given the risks of using animal serum for tissue engi- neering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimu- lating cells to engineer intervertebral disc tissues. Key words: platelet-rich plasma, growth factor, interver- tebral disc, extracellular matrix, proteoglycan, collagen. Spine 2006;31:959 –966 The relatively few cells that reside in the anulus fibrosus (AF) and nucleus pulposus (NP) maintain intervertebral disc (IVD) matrix metabolism homeostasis. Although the regulatory mechanisms are not well understood, nu- trition to the disc, cytokines, and growth factors are thought to play a role. 1,2 Recent studies have shown that growth factors can upregulate the production of matrix molecules and also stimulate the accumulation of matrix constituents during culture. 3,4 The stimulation of cell metabolism by growth factors positively affects the per- turbation of matrix maintenance even when the bio- chemical and biomechanical properties of disc tissues are compromised. 5 A recent study showed that the injection of a growth factor into normal IVDs 6 and the discs in a rabbit disc degeneration model induced an increase in disc height, 7 suggesting that growth factors may have a therapeutic use in the treatment of IVD degeneration. In those cases of IVD degeneration that exhibit advanced pathologic changes, apoptosis of IVD cells 8 and struc- tural defects, such as an anulus tear, can be observed. Several growth factors have been shown to positively modulate the metabolism of IVD cells. Transforming growth factor- (TGF-), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), and epi- dermal growth factor (EGF) stimulate cell proliferation and proteoglycan (PG) synthesis in vitro. 9 –12 PDGF also reduces the rate of cell apoptosis. 11 More recently, Ma- suda et al 3 and Yoon et al 4 showed that bone morpho- genetic protein-7 (BMP-7, also known as osteogenic pro- tein-1, OP-1) and BMP-2 both enhance PG metabolism in IVD cells. Furthermore, as cited above, the injection of BMP-7 was effective in increasing the disc height of de- generated rabbit IVDs. 7 Therefore, the application of growth factors via direct injection of recombinant proteins into the NP or the AF may be an effective therapeutic ap- proach in the treatment of IVD degeneration. For repair of larger defects, alternative therapeutic approaches, such From the Departments of *Orthopedic Surgery, †Biochemistry, and ‡Internal Medicine, Rush Medical College at Rush University Medical Center, Chicago, IL; §DePuy Biologics, DePuy Spine Incorporation, Raynham, MA; and Department of Orthopedic Surgery, Mie Univer- sity School of Medicine, Mie, Japan. Acknowledgment date: July 7, 2004. First revision date: April 29, 2005. Acceptance date: June 1, 2005. Supported by a research grant from DePuy Spine Inc. and in part by NIH grants 2-P50-AR39239 and 1-P01-AR48152. The device(s)/drug(s) that is/are the subject of this manuscript is/are not FDA-approved or approved by corresponding national agency for this indication. Corporate/Industry, Federal, and Institutional funds were received in support of this work. Although one or more of the author(s) has/have received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this manuscript, benefits will be directed solely to a research fund, founda- tion, educational institution, or other nonprofit organization which the author(s) has/have been associated. Address correspondence and reprint requests to Koichi Masuda, MD, 1735 W. Harrison St., Cohn 720, Chicago, IL 60612. E-mail: kmasuda@rush.edu 959