Purpose

Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold.

Purpose: Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological mechanism and full characterization of each individual stage through which the BM-MSC differentiate into adult chondrocytes is not yet understood. The physiological micro-environment of the chondrocytes is intensely hypoxic which triggers over-expression SLC2A proteins (GLUTs) in their membranes as a compensatory mechanism for energy production within the glycolytic cycle.

Method: We cultured and differentiated BM-MSC, and adult chondrocytes in hypoxic (5% O2 tension) and normoxic (20% O2) conditions. Within this cell populations we screened for the presence of the 12 GLUT genes as well as quantification of the variation of the 12 GLUTs gene translation by simple pcr and rt-pcr. The expression profile of the GLUT proteins was investigated using western blot analysis and immunohistochemistry. Functional characterization of the GLUTs expressed in the different cell populations was carried out by the means of radio-isotope labeled hexose fluxes done accordingly to the substrate specificity and kinetic properties particular to each SLC2A isoforms.

Results: Our data showed that the functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSC comprised an extensive expression of fructose-transporting GLUTs as opposed to the glucose-only transporting isoforms expression in normoxic BM-MSC. The flux data showed clear similarities in functional GLUT profiles between BM-MSC cultured in hypoxic conditions, adult chondrocytes. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-gluose, 3-orthomethyl-glucose and galactose) in these cellular populations under both hypoxic and normoxic conditions and in the presence and absence of Cytochalasin B (a GLUT1-specific inhibitor) showed that SLC2A class II transporters (GLUTs 5, 7, 9 and 11) play a more important role in the uptake of sugars by the normal hypoxic chondrocytes when compared to the ubiquitously-expressed GLUT1.

Conclusion: Use of this approach allows the correct culturing conditions to be identified that would select for those chondrocyte precursors from the total BM-MSC population that would have the best potential for producing viable articular cartilage. In addition, specific substrates for GLUTs isoforms could be used for physiologic, non-invasive and real time imaging of cartilage, BM-MSC and cartilage autograft by means of Positron Emission Tomography.

Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time.

Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with real time PCR, probing specifically for the genes responsible for the production of collagen I, collagen II, aggrecan, and GAPDH. This was done in duplicate.

Results: Collagen II gene expression was maintained over seven weeks in cells left at confluence but was decreased in passaged cells. Collagen I gene expression decreased over seven weeks in cells left at confluence, but remained the same in passaged cells. Aggrecan gene expression remained the same in both groups.

Conclusion: Current culture and expansion techniques that employ passaging (as used in clinical scenarios) result in significant alterations in gene expression that are inconsistent with the current definition of a “chondrocyte”. Culturing chondrocytes at confluence can produce gene expression more similar to native chondrocytes but even these cells have expression of collagen type I that should not be present in chondrocytes. The results of this study suggest that further investigation is required to develop chondrocyte culture and expansion techniques that minimize the de-differentiation of chondrocytes by maintaining collagen II gene expression and eliminating/preventing collagen I gene expression.

Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration.

Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes. Real time polymerase chain reaction was utilized to quantify and compare the essential markers, collagen II, collagen I and aggrecan, in the stem cell derived chondrocytes. The CD44 enriched and CD44 depleted populations were compared.

Results: The cells isolated possessed a cell morphology and surface antigen profile consistent with a MSC population. In addition, both experimental groups demonstrated multipotent ability. Real time PCR analysis of the chondrogenic cells demonstrated that the CD44 positive population expressed collagen II and aggrecan at a significantly higher level than the CD44 negative population.

Conclusion: To date no group has successfully identified a relationship between a MSC subpopulation and the multipotent progenitors responsible for generating cartilage. This work demonstrated that there are MSC sub-populations with different potential for chondrogenic expression and represents an important step towards identifying MSC subpopulations with enhanced cartilage formation potential.

Articular cartilage (AC) has a poor innate healing capacity following significant injury. Autologous chondrocyte implantation is a repair technique which utilises in vitro-expanded chondrocytes combined with a periosteal patch. The chondrocytes are enzymatically digested from arthroscopically harvested tissue at an initial surgery and expanded in monolayer culture prior to implantation at a second procedure. Unfortunately, in vitro expanded chondrocytes appear unable to retain their fundamental phenotype resulting in dedifferentiated cells which produce a matrix of inferior quality. This study compares the matrix-component gene expression profiles of chondrocytes in their native chondrons and through multiple divisions in monolayer culture. We hypothesised that there would be a rapid decline of matrix-component gene expression within a few cell replications in monolayer culture. The goal is to understand more fully the process of chondrocyte dedifferentiation and to compare matrix-component gene expression during cellular expansion in vitro.

Human AC was obtained from tissue donors and operative patients. A portion of the AC was stored at −80°C for use as a control while the remainder was homogenised and enzymatically digested with collagenase. The released cells were plated in monolayer culture and passaged (2:1) when they approached confluence. RNA was extracted from the frozen cartilage control and the passaged chondrogenic cell lines from which cDNA was generated. Real time PCR was performed with primers specific for collagen I, collagen II, aggrecan, and GAPDH. Gene expression was quantified and profiles from the cells in their native chondron and passaged cells (p0-p9) were compared.

Cells, when removed from the extra-cellular matrix and plated in monolayer, experienced an immediate upregulation of collagen I which persisted throughout all passages. In contrast, there was a stepwise decrease in collagen II with each successive passage until p8-p9 when the expression became undetectable. Aggrecan expression only decreased minimally as the cells were passaged.

Rapid dedifferentiation of monolayer cultured chondrocytes is a persistent barrier to AC tissue engineering including ACI. This study quantified the expression of relevant genes relating to AC generation and is an important first step to understanding cellular events, as alternative expansion techniques and cellular alternatives are sought.

This study was designed to examine the components of the MR image of the neurocentral junction (NCJ) and to explore the discrepancy between the age of closure of the NCJ as determined by anatomic and imaging studies. MR images of one hundred and fourteen porcine NCJs were correlated with anatomic and histologic sections. Whereas gross anatomic visualization did not reveal the NCJ site, MRI was sensitive for cartilage detection and accurately determined the age of NCJ closure although it overestimated the extent of closure. Based on this study, MRI characterization of the NCJ appears reliable and the NCJ cartilage does not close until adolescence.

This study examined the composition of the MR image of the neurocentral junction (NCJ) and the discrepancy between the age of closure of the NCJ as determined by anatomic and imaging studies.

MRI was sensitive for cartilage detection and accurately determined the age of NCJ closure (i.e. absence of cartilage on histologic examination).

Disparate NCJ development has been implicated as a potential cause of adolescent idiopathic scoliosis. Whereas autopsy studies have refuted this theory by suggesting that the NCJ closes before adolescence, MRI studies have resurrected this idea by suggesting later closure. MRI-histologic correlation suggests that the NCJ cartilage remains present until adolescence and therefore further exploration of the disparate growth hypothesis is required.

Gross anatomic visualization did not reveal the NCJ site, even after removal of the periosteum. In contrast, the presence or absence of an NCJ image correlated with the presence or absence of cartilage although MRI overestimated the extent of this cartilage.

Vertebrae were grossly examined for any evidence of the NCJ site. Sagittal and transverse MR images of one hundred and fourteen porcine NCJs in various stages of development (thirty-eight open, sixty-four closing, twelve closed) were correlated with anatomic and histologic sections acquired at the same position.

Funding: Edmonton Orthopaedic Research Association and University of Alberta Department of Radiology and Diagnostic Imaging