Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols.
Effective cryopreservation of articular cartilage (AC) could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. Vitrification (a form of cryopreservation) incorporates high concentrations of cryoprotectant agents (CPAs) and rapid cooling rates to preserve cells in suspended animation without detrimental ice formation. Effective vitrification requires high concentrations of CPAs within the cartilage matrix but the time-dependent toxicity of CPAs hinders their usefulness. The objective of this experiment was determine the CPA permeation parameters for four commonly used CPAs. This data will enable the use of mathematical models to develop novel vitrification procedures to preserve AC. We hypothesised that the time dependency of CPA permeation into intact AC can be determined by exposing AC to CPAs for specific times and then allowing the CPA to be removed into a known volume of PBS, the osmolarity of which is then measured. Full thickness 10mm diameter osteochondral dowels were harvested from the medial femoral condyles of sexually mature pigs. The dowels were randomly immersed in one of four CPAs (DMSO, propylene glycol, ethylene glycol, and glycerol) for various lengths of time (1–15 min). The cartilage was then immersed in 4ml of 1X PBS in a sealed container for twenty-four hours. The equilibrated solution was measured for osmolarity. The cartilage was weighed before and after treatment and this data was used to calculate the CPA concentration within the AC. This will be repeated in triplicate. Preliminary results (minimum n=2) indicated a marked difference in permeation for the four CPAs. Ethylene glycol had the most rapid permeation with almost complete permeation (84%) within 15 min. Conversely, glycerol had the least permeation (29%) after 15 min most of which occurred within the first minute. DMSO (63%) and propylene glycol (40%) had intermediate rates of permeation that gradually increased over time. Cryoprotectant agent permeation into intact porcine AC can be calculated using the method described in this study. This will allow us to successfully document the permeation kinetics of four commonly used CPAs within intact AC. This valuable data will markedly improve our ability to create novel vitrification solutions using mathematical models to add and remove CPAs to limit their toxic effects at high concentrations.
Current research strategies for studying articular cartilage (AC) repair include the observation of chondrocyte behaviour in monolayer cultures, the use of artificial matrices and animal models. Since AC relies on the diffusion of joint synovial fluid for nourishment, we hypothesised that it should be possible to develop a research model in which full-depth AC explants are maintained under established tissue culture conditions. Successful maintenance of explants for prolonged periods of time would represent a novel approach and provide a very powerful research tool to address a wide range of chondrocyte biology and matrix synthesis questions. The objective of the project was to examine the cell viability within an AC explant model maintained in tissue media. AC samples were obtained from the femoral condyles of total knee arthroplasty patients. Cylindrical dowels (10mm in diameter) were harvested from these samples. The dowels consisting of full-depth AC with several mm of subchondral bone attached were placed in tissue culture flask (T-25) containing 15mls of the respective culture media and maintained at 37oC in an incubator containing 5% CO2. Dowels were cultured in a variety of different media formulations (DMEM/F12, CGM (chondrocyte growth medium)) as well as PBS (phosphate buffered saline) which served as a negative control. AC chondrocyte viability was evaluated after five weeks. After having determined the best medium for cartilage maintenance, a second study with a broader range of end-points was undertaken. All dowels collected, rated 2/4 on the Outerbridge scale for osteoarthritis, and were then grown for zero, four, eight or twelve weeks in DMEM/F12 and CDM (chondrocyte differentiating medium). At each time interval, the dowels were evaluated for viability (live/dead stain), general morphology (trichrome stain), distribution of matrix proteins and proteoglycans (aggrecan, Types I and II collagen – immunofluorescence). After five weeks in PBS, there were no viable cells in the explant. Viability in the explants maintained in DMEM/F12 was 71% compared to 59.6% in the CGM treatment. The viability of the cells in the second study was 90% with DMEM/F12. After twelve weeks, the explant models stained well for general morphology and the distribution of proteoglycans and collagen was well maintained. To our surprise, the DMEM/F12 medium actually demonstrated the highest cell viability. Typically, AC requires joint motion to pressurise the synovial fluid into the matrix, which augments the transport of nutrients to the cells. Given that this study did not include any form fluid pressurization, it is surprising that such high cell viability was observed. This suggests that passive diffusion alone may provide adequate nourishment in this model system. In conclusion, the explant model for studying AC damage and repair examined in this research appears to be quite promising. This novel approach may serve as the foundation for subsequent research into new treatment strategies for AC injury.
Significant variation exists amongst surgeons regarding the post operative rehabilitation protocol after surgical repair of Achilles tendon ruptures. The objective of this study was to determine the effect of early weight bearing on the postoperative recovery after surgical repair of ruptured Achilles tendons. The hypothesis was that an early weight bearing routine does not increase the rate of re-rupture or other complications, and improves the quality of life by reducing discomfort of the rehabilitation phase and allowing an earlier return to work and sport. A prospective, randomised controlled trial of patients with Achilles tendon ruptures undergoing surgical repair was performed over a three year period concluding in April 2006. Specific inclusion and exclusion criteria were used to include one hundred and ten patients in the study at two centers involving twelve surgeons. Patients were randomised to either weight bearing or non weight bearing at the initial postoperative visit and compliance was monitored with a pressure sensor in the fixed-hinged ankle foot orthosis. Patients were assessed at six, twelve and twenty-six weeks postoperatively for 1) re-rupture rate, 2) strength of calf musculature 3) ankle range of motion, 4) complications, 5) return to sporting activities, and 6) return to work. In addition, health–related quality of life was measured using the SF-36 and AOFAS Ankle-Hindfoot Scale. Enrollment was completed as of May 2006 and six month data is over 90% completed. We are on course for <
10% loss to follow-up. Thus far, there have been no re-ruptures in either group. Definitive data analysis can only be performed once all one hundred and ten patients have completed their six month follow up. Once data analysis is completed, comparisons of patient satisfaction, strength, range of motion, complications, return to sport and return to work will be discussed. Early weight bearing after Achilles tendon repair did not increase the rate of re-rupture. Final data analysis will document any significant differences in the parameters measured. We believe this study will provide a definitive answer on the safety of early weight bearing after surgical repair of Achilles tendon ruptures.
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.
Cryopreserving agents (CPAs) can cryopreserve articular cartilage (AC) but their use is limited due to cellular toxicity. This study examined the time-dependent penetration of multiple CPAs into intact porcine AC. Porcine AC was immersed in CPAs for various amounts of time at three temperatures (4°C, 22°C, and 37°C). The results demonstrated an initial sharp rise in CPA concentration within the matrix for dimethyl sulfoxide and propylene glycol with maximum concentration after three to six hours. The trehalose and glucose concentration increased minimally even after twenty-four hours of exposure. The information from this study provides insight into the penetration kinetics of cryoprotectant agents into AC. This study examined the time-dependent penetration of cryoprotectant agents (CPAs) [dimethyl sulfoxide (DMSO), propylene glycol (PG), trehalose and glucose] into intact porcine articular cartilage (AC). Penetration of DMSO and PG into AC was rapid but time and temperature dependent while trehalose and glucose had poor penetration. The information gathered from this study can determine concentrations of CPAs within the cartilage matrix to create cryopreservation/vitrification solutions while minimizing toxicity. The results demonstrated there was a sharp rise in the CPA concentration within 15–30min exposure to DMSO and PG and the concentration peaked after three to six hours exposure at a concentration approximately 90% of the original concentration (6.5 molar). This was temperature dependent with slower penetration at lower temperatures. The trehalose and glucose had very poor penetration into the matrix at all temperatures, with a maximum penetration of 2% of the original concentration. Dowels of porcine AC (10mm diameter) were immersed in high concentration of each CPA for various amounts of time (0min, 15min, 30min, 60min, 3hr, 6hr, and 24hr) at three temperatures (4°C, 22°C, and 37°C). The cartilage was excised and the amount of cryoprotectant within the matrix determined. Successful cryopreservation of AC could improve clinical results of osteochondral allografting and provide a useful treatment alternative for large cartilage defects. However, successful cartilage cryopreservation is limited by chondrocyte death and matrix disruption due to inadequate CPA penetration.
Cryoprotectant toxicity has become more relevant because of increased use of high concentrations of cryoprotectants for vitrification of biologic tissues. A single toxicity model that integrates cryoprotectant concentration, time and temperature is essential to optimize the cryopreservation of tissues. The Weibull probabilistic distribution has been used in environmental toxicology research. This objective of this study was to fit the Weibull model to experimental data for chondrocyte recovery from articular cartilage exposed to various concentrations of dimethyl sulfoxide at different temperatures as a function of time. This study indicated that the Weibull model is an appropriate model to describe cryoprotectant toxicity to chondrocytes in articular cartilage. This study was designed to examine the toxicity of dimethyl sulfoxide (DMSO) on chondrocytes in porcine articular cartilage (AC) as a function of time, temperature and concentration. The Weibull model is suitable for modeling cryoprotectant toxicity in cartilage and can be further extended to other cellular and tissue systems. The model provides a simple method to predict toxicity and to assess the feasibility of cryopreservation protocols. The model proved to be a good fit for the entire data set of concentration, temperature and time, yielding an R2 value of 0.87 and a maximum discrepancy of 20% between the experimental data and the model. Estimates of the model’s parameters within a confidence interval of 95% were found to be: _=30±2, _=0.67±0.05, _C=0.38±0.03, _T=−2300±300 and _CT=700±100. Sliced porcine AC was exposed to DMSO (1, 3, 5, 6M) at different temperatures (0, 22, 37°C) for various durations. Cellular viability was determined by membrane integrity stains. Experimental data for chondrocyte recovery was fit to the global Weibull probabilistic distribution model using SPSS SigmaPlot 2000 to estimate the five parameters. A model integrating concentration, time, and temperature of exposure is required to optimize addition and removal protocols of high concentrations of cryoprotectant for cryopreservation. The Weibull distribution is a simple and flexible model used to describe similar processes. In the current study, chondrocyte viability decreased with increased concentration, temperature and time of exposure. The model indicated a significant interaction between the toxic effects of concentration and temperature.
We compared the radiographs, clinical outcomes, and complications of two techniques used for treatment of unstable distal radius fractures in young adults. Fifty patients were randomized to percutaneous pinning or augmented external fixation. At one year follow-up, the external fixator did not improve the parameters of radial length, radial angulation or volar tilt. Reduction of intra-articular steps was slightly improved. No differences in DASH scores or functional outcomes were observed, but more complications were noted with the fixator. While articular restoration can be slightly improved with use of the external fixator, a higher incidence of complications and patient dissatisfaction was noted. Two common techniques for treatment of unstable distal radius fractures in young adults include percutaneous pinning combined with plaster cast, and application of an external fixator, frequently with adjunctive pinning. The objective of this study was to: 1. To compare the short and mid-term radiographic and clinical outcomes of these two common fixation techniques. 2. To compare the complications of the two techniques. Fifty patients (<
65 yrs) with unstable fractures of the distal radius were recruited. Patients were randomized pre-operatively to percutaneous pinning or external fixation. All surgery was performed by one of three surgeons. Patients were followed for one year with radiographs and an independent clinical exam including DASH questionnaires. 86% of fractures were AO classification C2 or C3, with an equal distribution of all types in both treatment groups. Use of an external fixator did not improve the parameters of radial length, radial angulation or volar tilt. However, reduction of intra-articular steps was slightly improved with its’ use. No differences in mean DASH scores, total ROM or grip strength were observed. More pin complications were noted with the fixator, and all three patients diagnosed with RSD received external fixation. While external fixation represents a popular first line treatment for unstable distal radius fractures, this study suggests that similar gross radiographic and clinical results can be obtained with percutaneous pinning. While articular restoration can be slightly improved with use of the external fixator in highly comminuted fractures, this must be balanced by a higher incidence of complications and patient dissatisfaction.
