Kelly Leslie

Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat.

Study Design. Work presented here used a small animal model to illustrate the feasibility of autologous disc cell implantation. Objectives. To develop a small animal model for autologous disc cell implantation. Summary of the Background Data. The use of autologous disc cells in the potential treatment of disc degeneration offers attractive possibilities for novel therapies. Results are presented on the use of the sand rat (Psammomys obesus), a small rodent that spontaneously develops disc degeneration during aging, in experimental studies in which cells were harvested from a lumbar intervertebral disc, expanded in monolayer tissue culture, labeled with agents that allow subsequent immunolocalization of these cells, and implanted in a second disc site of the donor animal. Methods. Tissue culture, disc surgery, histology, and immunocytochemistry were used. Cells were either engrafted in a bioresorbable carrier tested for cell compatibility or injected into the recipient disc. Results were assessed with radiographic examination of the implantation site and with histology and immunocytochemistry. Conclusion. Data from 15 animals were obtained with engraftment resident in the animal for up to 33 weeks. Immunocytologic identification of engrafted cells showed that they integrated into the disc and were surrounded by normal matrix at time points up to 8 months postengraftment. Engrafted cells exhibited either a spindle-shaped morphology in the annulus or a rounded chondrocyte-like morphology in the nucleus. Although technically challenging, the authors’ experience showed that autologous disc cell implantation can be successful and that the sand rat is a valuable model for autologous disc cell studies.

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.

Cellular, but not matrix, immunolocalization of SPARC in the human intervertebral disc: Decreasing localization with aging and disc degeneration

Study Design. Human intervertebral disc anulus tissue was obtained in a prospective study of immunolocalization of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin). Experimental studies were approved by the authors’ Human Subjects Institutional Review Board. Discs were obtained from surgical specimens and from control donors. Objectives. To determine whether SPARC could be detected in the disc with immunohistochemistry and to determine the incidence of SPARC-positive cells. Summary of the Background Data. SPARC is a glycoprotein that has an important role in modulating interactions between cells and matrix. It influences remodeling, collagen fibrillogenesis, metalloproteinase expression, and cytokine expression. Little is known about SPARC in the disc, and one previous study reported the absence of its immunolocalization in fetal and adult disc tissue. Methods. Eight normal human discs from subjects aged newborn to 10 years, and 11 disc specimens from control donors or surgical patients aged 15to 76 years were examined for immunolocalization of SPARC. Anulus cells were also tested for the presence of SPARC in vitro in monolayer or three-dimensional agarose culture. Results. In discs of subjects aged newborn to 0.19 years, SPARC was present in all cells in the outer anulus, in 76.4% of inner anulus cells, and 76.0% of nucleus cells. Localization was significantly lower in anulus cells of study participants aged 4.7 to 76 years (66.7%, P = 0.04). Anulus cells cultured in agarose or monolayer showed positive localization in all cells. Conclusions. Findings show decreased presence of SPARC in disc cells of older subjects with disc degeneration and point to the importance of future studies designed to elucidate the unrecognized role of SPARC in disc remodeling, aging, and degeneration.

Expression and localization of estrogen receptor-β in annulus cells of the human intervertebral disc and the mitogenic effect of 17-β-estradiol in vitro

BackgroundRecent evidence suggests that estrogens exert effects in different tissues throughout the body, and that the estrogen receptor β (ERβ) may be important for the action of estrogen (17-β-estradiol) on the skeleton. The cellular localization of ERβ in the human intervertebral disc, however, has not yet been explored.MethodsHuman disc tissue and cultured human disc cells were used for immunocytochemical localization of ERβ. mRNA was isolated from cultured human disc cells, and RT-PCR amplification of ERβ was employed to document molecular expression of this receptor. Cultured human disc cells were tested to determine if 17-β-estradiol stimulated cell proliferation.ResultsIn this report data are presented which provide evidence for ERβ gene expression in human intervertebral disc cells in vivo and in vitro. Culture of annulus cells in the presence of 10-7 M 17-β-estradiol significantly increased cell proliferation.ConclusionsThese data provide new insight into the biology of cells in the annulus of the intervertebral disc.

Clinical and demographic prognostic indicators for human disc cell proliferation in vitro: pilot study.

Study Design. Human anulus cells were cultured under control and experimental conditions to study associations between proliferation and clinical–demographic features of subjects from which cells were obtained. Statistical multiple regression analyses were applied to develop mathematic models relating proliferation to age, gender, Thompson score (denoting stage of disc degeneration), and status (control donor [postmortem]; surgical patient). Objectives. To identify the effect of donor characteristics on proliferative capacities of human disc cells. Summary of Background Data. As therapeutic options for disc degeneration increase, novel biologic options are important future considerations. Little is known about the influence of clinical–demographic features on cell proliferation. Methods. Anulus cells were studied in two designs: 1) Cells from 12 individuals were grown in monolayer with 50 ng/mL interleukin growth factor-1 (IGF-I), 100 ng/mL insulin, or control conditions. 2) Cells from nine individuals were grown in three-dimensional culture with 10 ng/mL IGF-I or control conditions. Cell proliferation data and data on age, gender, Thompson score, and status were collected. Standard statistical analyses were used to develop correlation models. Results. Data from monolayer experiments produced significant models fitting proliferation in the presence of low serum, 50 ng/mL IGF-I, or insulin, with age, gender, Thompson score, and status (respective R2: 0.827, 0.680, 0.850). Three-dimensional cultures exposed to 10 ng/mL IGF-I resulted in proliferation that correlated in a significant negative manner with Thompson score (r = −0.798). Conclusions. Clinical–demographic prognostic indicators may help predict levels of proliferation. Greater age, greater disc degeneration, female gender, and surgical derivation had deleterious effects on proliferation potential in this model.

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.

Human intervertebral disc cells

Abstract Purpose of study: The objective of this study was to investigate how human disc cells in vitro attached, proliferated, produced extracellular matrix (ECM) and expressed selected ECM genes within various three-dimensional (3D) carrier materials. Methods used: Studies were approved by the human subjects institutional review board. Specimens were obtained from surgical procedures performed on individuals with herniated discs. Specimens were transported immediately and cells established using published methods. Control donor discs were obtained by means of the NCI Cooperative Human Tissue Network. Cells were expanded in culture and tested for attachment, proliferation and ECM production in collagen sponges, collagen gel, agarose, alginate or fibrin gel formulations after 10 days of culture. Reverse transcriptase polymerase chain reaction (RT-PCR) in situ hybridization was used to assess ECM gene expression for Types I and II collagen, aggrecan and chondroitin sulfate. Histologic assessment included routine staining and immunohistochemistry. Cell cultures from the annulus were established from 29 individuals (mean age, 45.3 years; range, 26 to 73 years; 17 males, 12 females). Distribution of Thompson grades were grade II, 5 individuals; grade II, 12; grade IV, 12. Twenty-four sites were lumbar, four were cervical and one was lower thoracic. of findings: Collagen sponges provided the best microenvironment for disc cell ECM production ( Fig. 1 Download : Download high-res image (635KB) Download : Download full-size image Fig. 1 . , far left) and gene expression ( Fig. 1, A , Type I collagen; B, Type II collagen: C, chondroitin sulfate; D, aggrecan). Although collagen gels supported cell growth, they did not result in either abundant ECM production or ECM gene expression as shown by RT-PCR in situ hybridization. Growth, ECM production and gene expression in alginate, agarose and fibrin microenvironments were also inferior. Relationship between findings and existing knowledge: Cell technologies and tissue engineering techniques have opened new therapeutic possibilities for use of autologous cells in clinical orthopedic applications. For potential biologic intervertebral disc therapies, fundamental questions on how human disc cells interact with and grow in potential cell carriers are unexplored. Overall significance of findings: Results provide novel data on disc cell survival and gene expression within diverse microenvironments. Collagen in a 3D sponge microenvironment proved superior in terms of cell survival, ECM production and ECM gene expression as reflected in assessment of Types I and II collagen, aggrecan and chondroitin sulfate. Disclosures: No disclosures. Conflict of interest: Helen Gruber, and Edward Hanley, Jr., grant research support: North American Spine Society