Intervertebral disc cells as competent phagocytes in vitro: implications for cell death in disc degeneration

INTRODUCTION: Apoptosis has been reported to occur in the intervertebral disc. Elsewhere in the body, apoptotic cells are cleared from the system via phagocytosis by committed phagocytes such as macrophages, reducing the chance of subsequent inflammation. These cells, however, are not normally present in the disc. We investigated whether disc cells themselves can be induced to become phagocytic and so have the ability to ingest and remove apoptotic disc cells, minimising the damage to their environment. METHOD: Bovine nucleus pulposus cells from caudal intervertebral discs were grown in culture and exposed to both latex particles (which are ingested by committed phagocytes) and apoptotic cells. Their response was monitored via microscopy, including both fluorescent and video microscopy, and compared with that seen by cell lines of monocytes/macrophages (THP-1 and J774 cells), considered to be committed phagocytes, in addition to a nonmacrophage cell line (L929 fibroblasts). Immunostaining for the monocyte/macrophage marker, CD68, was also carried out. RESULTS: Disc cells were able to ingest latex beads at least as efficiently, if not more so, than phagocytic THP-1 and J774 cells. Disc cells ingested a greater number of beads per cell than the committed phagocytes in a similar time scale. In addition, disc cells were able to ingest apoptotic cells when cocultured in monolayer with a UV-treated population of HeLa cells. Apoptotic disc cells, in turn, were able to stimulate phagocytosis by the committed macrophages. CD68 immunostaining was strong for THP-1 cells but negligible for disc cells, even those that had ingested beads. CONCLUSION: In this study, we have shown that intervertebral disc cells are capable of behaving as competent phagocytes (that is, ingesting latex beads) and apoptotic cells. In terms of number of particles, they ingest more than the monocyte/macrophage cells, possibly due to their greater size. The fact that disc cells clearly can undergo phagocytosis has implications for the intervertebral disc in vivo. Here, where cell death is reported to be common yet there is normally no easy access to a macrophage population, the endogenous disc cells may be encouraged to undergo phagocytosis (for example, of neighbouring cells within cell clusters)

Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology

Autologous chondrocyte implantation is being used increasingly for the treatment of cartilage defects. In spite of this, there has been a paucity of objective, standardised assessment of the outcome and quality of repair tissue formed. We have investigated patients treated with autologous chondrocyte implantation (ACI), some in conjunction with mosaicplasty, and developed objective, semiquantitative scoring schemes to monitor the repair tissue using MRI and histology. Results indicate repair tissue to be on average 2.5 mm thick. It was of varying morphology ranging from predominantly hyaline in 22% of biopsy specimens, mixed in 48%, through to predominantly fibrocartilage, in 30%, apparently improving with increasing time postgraft. Repair tissue was well integrated with the host tissue in all aspects viewed. MRI scans provide a useful assessment of properties of the whole graft area and adjacent tissue and is a noninvasive technique for long-term follow-up. It correlated with histology (P = 0.02) in patients treated with ACI alone

Bovine explant model of degeneration of the intervertebral disc

BACKGROUND: Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. METHODS: Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue). RESULTS AND CONCLUSION: The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies

Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation

OBJECTIVE: Autologous chondrocyte implantation (ACI) is a form of tissue engineering that is being used increasingly to treat damaged articular cartilage. What happens at the graft site subsequent to the transplantation of chondrocytes beneath a periosteal flap has largely remained a matter of conjecture. We examined biopsy samples from the graft site using a panel of specific antibodies to investigate the cellular mechanisms involved and to determine whether remodeling of the matrix occurs. METHODS: Ten full-depth core biopsy samples were obtained from patients who had undergone ACI 9-30 months previously (ages 28-53 years), in addition to 6 "control" biopsy samples. Cryosections were evaluated by standard histologic examination using polarized light and immunohistochemistry. Antibodies specific for type II collagen (CIIC1) were used, as well as antibodies against the C-propeptide of type II collagen (R160) and its denaturation product (Col2-3/4m), as indicators of anabolism or catabolism. In addition, antibodies to the matrix proteinase-generated neoepitopes of the aggrecan core protein were used to demonstrate either aggrecanase (BC-3 and BC-13) or matrix metalloproteinase (MMP) (BC-4 and BC-14) activity. RESULTS: All biopsy samples stained for type II collagen, even in areas of fibrocartilaginous morphology. There was evidence of newly synthesized type II collagen in addition to denatured collagen. MMP and aggrecanase activity on the proteoglycan population was evident, with aggrecanase being more active in fibrocartilaginous areas. CONCLUSION: The findings of this study indicate that ACI is capable of not only cartilage repair but, in some cases, regeneration. This may be achieved by the turnover and remodeling of an initial fibrocartilaginous matrix via enzymatic degradation and synthesis of newly formed type II collagen

Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation

OBJECTIVE: Autologous chondrocyte implantation (ACI) is a form of tissue engineering that is being used increasingly to treat damaged articular cartilage. What happens at the graft site subsequent to the transplantation of chondrocytes beneath a periosteal flap has largely remained a matter of conjecture. We examined biopsy samples from the graft site using a panel of specific antibodies to investigate the cellular mechanisms involved and to determine whether remodeling of the matrix occurs. METHODS: Ten full-depth core biopsy samples were obtained from patients who had undergone ACI 9-30 months previously (ages 28-53 years), in addition to 6 "control" biopsy samples. Cryosections were evaluated by standard histologic examination using polarized light and immunohistochemistry. Antibodies specific for type II collagen (CIIC1) were used, as well as antibodies against the C-propeptide of type II collagen (R160) and its denaturation product (Col2-3/4m), as indicators of anabolism or catabolism. In addition, antibodies to the matrix proteinase-generated neoepitopes of the aggrecan core protein were used to demonstrate either aggrecanase (BC-3 and BC-13) or matrix metalloproteinase (MMP) (BC-4 and BC-14) activity. RESULTS: All biopsy samples stained for type II collagen, even in areas of fibrocartilaginous morphology. There was evidence of newly synthesized type II collagen in addition to denatured collagen. MMP and aggrecanase activity on the proteoglycan population was evident, with aggrecanase being more active in fibrocartilaginous areas. CONCLUSION: The findings of this study indicate that ACI is capable of not only cartilage repair but, in some cases, regeneration. This may be achieved by the turnover and remodeling of an initial fibrocartilaginous matrix via enzymatic degradation and synthesis of newly formed type II collagen

(a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control)

(b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.<p><b>Copyright information:</b></p><p>Taken from "Bovine explant model of degeneration of the intervertebral disc"</p><p>http://www.biomedcentral.com/1471-2474/9/24</p><p>BMC Musculoskeletal Disorders 2008;9():24-24.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266744.</p><p></p

The profile from outer annulus to the central nucleus pulposus of (a) water and (b) GAG contents of fresh normal, bovine disc (i

E. not injected with enzyme or cultured).<p><b>Copyright information:</b></p><p>Taken from "Bovine explant model of degeneration of the intervertebral disc"</p><p>http://www.biomedcentral.com/1471-2474/9/24</p><p>BMC Musculoskeletal Disorders 2008;9():24-24.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266744.</p><p></p