Frances Henson

A major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage. Human primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes. The chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600-fold, p < 0.0001) and SRY box 9 (SOX9 30-fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation. The results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.

Augmented microfracture techniques use growth factors, cells and/or scaffolds to enhance the healing of microfracture treated cartilage defects. This study investigates the effect of delivering recombinant human fibroblastic growth factor 18 (rhFHF18, Sprifermin) via a collagen membrane on the healing of a chondral defect treated with microfracture in an ovine model. 8mm diameter chondral defects were created in the medial femoral condyle of 40 sheep (n = 5/treatment group). Defects were treated with microfracture alone, microfracture + intra-articular rhFGF18 or microfracture + rhFGF-18 delivered on a membrane. Outcome measures included mechanical testing, weight bearing, International Cartilage Repair Society repair score, modified O'Driscoll score, qualitative histology and immunohistochemistry for types I and II collagen. In animals treated with 32μg rhFGF-18 + membrane and intra-articularly there was a statistically significant improvement in weight bearing at 2 and 4 weeks...

The failure rate of rotator cuff repair is high. Regenerative techniques using material scaffolds, stem cells, and growth factors help augment repair and regenerate tissue. We reviewed the literature of various regenerative techniques in terms of (1) enhancing the repair process, (2) tissue regeneration, (3) mechanical strength, and (4) clinical outcome.

Microfracture is a common cartilage repair procedure. Strategies to improve healing post-microfracture include the use of growth factors to enhance hyaline cartilage production. This study investigates the effect of intra-articular recombinant human fibroblastic growth factor 18 (rhFHF18) on the healing of a chondral defect treated with microfracture in an ovine model. Chondral defects (8 mm diameter) were created in the medial femoral condyle of 80 sheep (n = 16/treatment group). Defects were treated with microfracture alone or microfracture + intra-articular rhFGF-18 (administered either as one or two cycles of 3× weekly injections). Outcome measures included mechanical testing, macroscopic International Cartilage Repair Society repair score, modified O'Driscoll histology score, qualitative histology, and immunohistochemistry for types I, II, and VI collagen. In treated animals, there was a statistically significant improvement in ICRS tissue repair score and tissue infill score, in the modified O'Driscoll score between control and 1 cycle of rhFGF-18 at 6 m, and in the cartilage repair score and structural characteristic score between the control and both rhFGF-18 groups at 6 m. There was no evidence of degeneration of adjacent cartilage in the rhFGF-18 treated cartilage. The increase in hyaline cartilage-like tissue formed in the microfracture + rhFGF-18 treated groups indicates that rhFGF-18 potentiates the formation of hyaline cartilage repair following microfracture.

Chondrocytes within articular cartilage respond to the mechanical stresses associated with normal joint loading via a series of signalling pathways. Specific biomolecules, such as nitric oxide (NO), have been implicated in these mechanotransduction processes. It has been shown that the synthesis of NO can be inhibited by dynamic compressive strain of chondrocytes in vitro which, in turn, leads to an up-regulation of specific metabolic parameters. Chondrocytes isolated from different joint locations and seeded in agarose constructs respond in a distinct manner to the application of dynamic compression. Chondrocytes were isolated separately from the equine patella groove and the femoral condyle, representing high loaded areas (HLA) and low loaded areas (LLA), respectively, of 6 specimens of different ages. The cells were seeded in agarose constructs and cultured either in an unstrained state or strained under dynamic loading at 1 Hz for 48 h. The synthesis of nitric oxide (NO), proteoglycan synthesis and chondrocyte proliferation were assessed. Equine chondrocytes were found to synthesise significant basal levels of NO, regardless of topographical origin or age of tissue. Marked differences in both proteoglycan synthesis and cell proliferation were, however, revealed between the 2 chondrocyte subpopulations. Dynamic compression inhibited NO synthesis but significant alterations in proteoglycan synthesis and cell proliferation were apparent in a minority of cases. The differential response of the subpopulations of chondrocytes derived from the HLA and LLA provides a potential mechanism which enables the biomechanical demands of differing joint regions to be maintained.

Sterile surgical marker pens are commonly used in cartilage repair surgery to aid in the placement of periosteal patches or collagen membranes in autologous chondrocyte implantation. To investigate the effects that methylene blue and crystal violet marker pen ink have on human chondrocytes when cultured on collagen membranes in vitro. Controlled laboratory study. Human chondrocytes were applied to Chondro-Gide collagen membranes at a volume of 12 million cells. In the first experiment, 2 sterile marker pens, one containing methylene blue and the other crystal violet inks, were used to mark membranes immediately before the addition of cells. In the second experiment, the same marker pens marked the membranes after 7 days of cell culture. In each experiment, 3 groups of membrane were tested for each pen. Group A consisted of no ink mark, group B had only the uppermost "smooth" layer marked, and group C had the lower "porous" layer marked. All membranes were then cultured in standard growth media for 24 hours. Cell viability was assessed at 24 hours on all membranes using a live/dead-cell viability assay. Cell viability was quantified with florescent microscopy with mean percentage of live cells in each marker pen group compared with control membranes using the Student t test (P < .05). Control membranes (group A) with no ink showed cell viability approaching 100%. A statistically significant reduction in cell viability with both methylene blue (23.1%; P < .0001) and crystal violet (18.9%; P < .0001) was found adjacent to the ink mark on the smooth side (group B) and on the porous side remote from the ink (group C) in both experiments (<30%; P < .0001). A reduction in cell viability was noted on the smooth side remote from the ink mark but did not reach statistical significance. Marked cell death was seen with both dyes (<15%; P < .0001) adjacent to the ink on the porous side. Chondrocyte viability is significantly reduced when cells are cultured in vitro on collagen membranes marked with methylene blue and crystal violet pen ink. Surgeons should be aware of the potential negative effect of marker pens in cell-based therapies.