Study Design Responses of mesenchymal stem cells (MSCs) from 2 age groups was analyzed under chemical conditions representative of the intervertebral disc (IVD) (low glucose levels, acidic pH, high osmolarity, and combined conditions). Objective To determine the microenvironmental conditions of the IVD that are critical for MSC-based tissue repair and to determine whether MSCs from different age groups respond differently. Summary of Background Data MSCs offer promise for IVD repair, but their potential is limited by the harsh chemical microenvironment in which they must survive. Methods MSCs were isolated from bone marrow from mature (4–5 month old) and young (1 month old) rats and cultured in monolayer under IVD-like glucose, osmolarity, and pH conditions as well as under a combination of these conditions and under standard media conditions for 2 weeks. The response of MSCs was examined by measuring gene expression (real-time RT-PCR), proliferation (MTT assay), and viability (fluorescence staining). Results Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen-1 expression and caused a small increase in proliferation. In contrast, IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions strongly decreased proliferation and expression of matrix proteins, with more pronounced effects for osmolarity. Combining these 3 conditions also resulted in decreased proliferation, and gene expression of matrix proteins, demonstrating that osmolarity and pH dominated the effects of glucose. Both age groups showed a similar response pattern to the disc microenvironment. Conclusion IVD repair using MSCs requires increased knowledge of MSC response to the chemical microenvironment. IVD-like low glucose enhanced matrix biosynthesis and maintained cell proliferation whereas IVD-like high osmolarity and low pH conditions were critical factors that reduced biosynthesis and proliferation of young and mature MSCs. Since osmolarity decreases and acidity increases during degeneration, we speculate that pH may be the major limitation for MSC-based IVD repair.

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The intervertebral disk is routinely subjected to compressive loads that alter with posture and muscle activity and can produce pressures > 2 MPa in human lumbar disks in vivo (A. Nachemson and G. Elfstrom. Scand. J. Rehabil. Med. 2, Suppl. 1:1-40, 1979; A. Nachemson and J. M. Morris. J. Bone Jt. Surg. Am. Vol. 46A: 1077-1092, 1964). We measured the effect of load on hydrostatic pressures in bovine caudal disks. With increase in applied load, pressure increased linearly in the nucleus and inner annulus. The resting pressure measured after slaughter (0.19 +/- 0.05 MPa) and the pressure at failure (34 MPa, estimated from the vertebrae/disk segment failure load of 7,430 +/- 590 N) define the limits that can occur in vivo. Because hydrostatic pressure influences matrix synthesis in articular cartilage, we have examined the effects of pressures in the range 1-10 MPa applied for 20 s or 2 h on proteoglycan synthesis in bovine caudal and human lumbar intervertebral disks in vitro. In the nucleus pulposus and inner annulus of bovine disks, application of hydrostatic pressure in the range of 1-7.5 MPa for only 20 s stimulated matrix synthesis over the following 2 h at atmospheric pressure. The maximum stimulation in the bovine disks was seen in the inner annulus after application of 2.5 MPa, where proteoglycan synthesis rates doubled. Exposure to 2.5 MPa also stimulated synthesis in the nucleus pulposus of human disks taken at surgery, whereas 7.5 MPa inhibited synthesis in five out of six specimens. With 2-h continuous exposure to the same levels of pressure, no stimulation was seen in the nucleus of bovine disks, and significant stimulation was only observed at 5.0 MPa in the inner annulus. Exposure to 10 MPa for either 20 s or 2 h inhibited proteoglycan synthesis in these regions of the disks. In contrast, in the outer annulus, where loading does not lead to a rise in hydrostatic pressure in vivo, there was no significant response to hydrostatic pressure over the range of 1-10 MPa in bovine or human disks.

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