Dose-injury relationships for cryoprotective agent injury to human chondrocytes.

Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose-injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy µm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me(2)SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose-injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me(2)SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me(2)SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.

Cryoprotective agent toxicity interactions in human articular chondrocytes.

BACKGROUND: Vitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ≥6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes. METHODS: Chondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solution's toxicity. RESULTS: Multiple-CPA solutions were significantly less toxic than single-CPA solutions (P<0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO-EG-Gy-Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs. CONCLUSIONS: Multiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO-EG-Gy-Fm combination solution.

The effect of pulsed electromagnetic fields on chondrocyte morphology.

Osteoarthritis is a debilitating joint disease where the articular cartilage surface degrades and is unable to repair itself through natural processes. Chondrocytes reside within the cartilage matrix and maintain its structure. We conducted in vitro experiments to investigate the morphological response of cultured human chondrocytes under different pulsed electromagnetic field (PEMF) conditions. In the control experiments, cultured chondrocytes attached to the bottom of a culture dish typically displayed either a stellate or spindle morphology with extended processes. Experimental chondrocyte cultures were placed in a Helmholtz coil to which a ramp waveform was applied. Exposure to PEMFs caused the chondrocytes to retract their processes, becoming spherical in shape. This change in morphology followed a progression from stellate to spindle to spherical. These morphological changes were reflected in an average reduction of 30% in the surface contact area of the chondrocytes to the culture dish. Understanding the mechanisms by which PEMFs affect the morphology of chondrocytes will help lead to new treatments for osteoarthritis.

The development of osteoblasts from stem cells to supplement fusion of the spine during surgery for AIS.

Surgical correction in severe cases of AIS is often hampered by insufficient autograft bone to facilitate the fusion. The development of other sources of bone generating cells would greatly enhance the surgical. Bone marrow derived stem cells were harvested from femoral reaming during total hip arthroplasty for the purpose of differentiating into osteoblasts. Stem cells were isolated from the marrow and successfully differentiated into three cell lines (osteoblasts, chondrocytes and adipocytes) to confirm multilineage potential. Osteoblasts were developed from the stem cells and demonstrated the ability to be cultured to possibly provide a source of bone generating cells to augment surgical fusions. It is anticipated that the addition of osteoblasts created from stem cells (combined with appropriate matrix) will have significant influence on the success of AIS surgery through improvement of bone fusion.

Acute Osteochondral Fractures in the Lower Extremities - Approach to Identification and Treatment.

Chondral and osteochondral fractures of the lower extremities are important injuries because they can cause pain and dysfunction and often lead to osteoarthritis. These injuries can be misdiagnosed initially which may impact on the healing potential and result in poor long-term outcome. This comprehensive review focuses on current pitfalls in diagnosing acute osteochondral lesions, potential investigative techniques to minimize diagnostic errors as well as surgical treatment options. Acute osteochondral fractures are frequently missed and can be identified more accurately with specific imaging techniques. A number of different methods can be used to fix these fractures but attention to early diagnosis is required to limit progression to osteoarthritis. These fractures are common with joint injuries and early diagnosis and treatment should lead to improved long term outcomes.

Intramatrix events during cryopreservation of porcine articular cartilage using rapid cooling.

Cryopreservation of articular cartilage may improve long-term transplantation results if cell and matrix integrity can be maintained. This study examined intramatrix events in intact porcine articular cartilage that occurred during a rapid-cooling technique with various concentrations of dimethyl sulfoxide (DMSO) (1, 3, 5, 6 and 7 M). Thermocouples were inserted into the solution and in the cartilage matrix to record the temperature during rapid cooling. In addition, scanning electron microscopy of freeze-substituted samples was performed and quantitatively evaluated for the areas representing ice in the matrix. The results of this study showed that low concentrations of DMSO resulted in the largest temperature gradient between the matrix and the surrounding solution, which occurred near the freezing point of the cryoprotectant solution. At higher concentrations of DMSO, the peak temperature gradient occurred near the glass transition temperature. The temperature measurements suggested that a significant amount of ice formed within the matrix at lower DMSO concentrations. At higher DMSO concentrations that resulted in vitrification of the external solution, there was evidence of some ice in the matrix. The scanning electron micrographs demonstrated significantly more matrix disruption (likely due to ice formation) (P<0.02) in the lower DMSO concentrations (1 and 5 M) while the 6 M DMSO concentration demonstrated minimal matrix disruption. Cryopreservation of articular cartilage with a rapid-cooling technique and high concentrations of DMSO resulted in partial vitrification of the matrix and significantly less matrix disruption. It appears that successful cryopreservation of viability and function in articular cartilage will require high concentrations of cryoprotectants and rapid cooling.

Cryopreservation of intact human articular cartilage.

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (-10, -20, -30, -40 degrees C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.

Arthroscopic reconstruction of the anterior cruciate ligament with patellar-tendon autograft and interference screw fixation. The results at seven years.

Deficiency of the anterior cruciate ligament (ACL) is a common disorder which can lead to changes in lifestyle. We followed 59 patients who had had arthroscopic reconstruction of the ACL using a central-third patellar-tendon autograft for seven years to assess the long-term effectiveness of recent advances in reconstruction of the ACL. The standard criteria for evaluation of the International Knee Documentation Committee, the Lysholm knee score and measurements using the KT 1000 arthrometer all showed satisfactory results. Deterioration in the clinical performance after seven years was associated with osteoarthritic changes and correlated with chronic ligament injuries and meniscectomy. There were three traumatic and three spontaneous ruptures. We believe that the procedure can be successful, but remain concerned about failure of the graft and osteoarthritis. The results raise questions about the best time to operate and suggest that early surgery may reduce the risk of osteoarthritis.

Operative reconstruction after transverse rupture of the tendons of both peroneus longus and brevis. Surgical reconstruction by transfer of the flexor digitorum longus tendon.

Rupture of the tendons of both peroneus longus and peroneus brevis results in considerable disability. We have performed transfer of flexor digitorum longus (FDL) to peroneus brevis in two patients with lateral instability of the hindfoot due to chronic transverse tears of both tendons for which end-to-end repair was not possible. Both patients had excellent function when reviewed after eight and six years, respectively, with no symptoms. CT showed a normal appearance of the FDL in both patients, but the peroneal muscles looked abnormal. Transfer of the FDL provides a reliable solution to lateral instability of the hindfoot resulting from loss of function of both peronei.

Validation and reproducibility of computerised cell-viability analysis of tissue slices.

BACKGROUND: The identification of live cells using membrane integrity dyes has become a frequently used technique, especially with articular cartilage and chondrocytes in situ where tissue slices are used to assess cell recovery as a function of location. The development of a reproducible computerised method of cell evaluation would eliminate many variables associated with manual counting and significantly reduce the amount of time required to evaluate experimental results. METHODS: To validate a custom computerised counting program, intra-person and inter-person cell counts of nine human evaluators (three groups - unskilled, novice, and experienced) were compared with repeated pixel counts of the custom program on 15 digitised images (in triplicate) of chondrocytes in situ stained with fluorescent dyes. RESULTS: Results indicated increased reproducibility with increased experience within evaluators [Intraclass Correlation Coefficient (ICC) range = 0.67 (unskilled) to 0.99 (experienced)] and between evaluators [ICC = 0.47 (unskilled), 0.85 (novice), 0.93 (experienced)]. The computer program had perfect reproducibility (ICC = 1.0). There was a significant relationship between the average of the experienced evaluators results and the custom program results (ICC = 0.77). CONCLUSIONS: This study demonstrated that increased experience in cell counting resulted in increased reproducibility both within and between human evaluators but confirmed that the computer program was the most reproducible. There was a good correlation between the intact cell recovery determined by the computer program and the experienced human evaluators. The results of this study showed that the computer counting program was a reproducible tool to evaluate intact cell recovery after use of membrane integrity dyes on chondrocytes in situ. This and the significant decrease in the time used to count the cells by the computer program advocate its use in future studies because it has significant advantages.

Chondrocyte recovery in cryopreserved porcine articular cartilage after bone carrier alteration.

In order to investigate the consequences on the distribution of cell recovery through a cross-section of articular cartilage, the pathway for ice nucleation and diffusion of water and solutes in porcine osteochondral tissue was altered by drilling a 2mm diameter hole through the subchondral bone to the base of the cartilage. Samples equilibrated with 1M dimethyl sulfoxide were cooled at 1 C/min to -30 degrees C then stored in liquid nitrogen. A significant increase in chondrocyte recovery was documented when compared to samples cryopreserved without holes (48.3 percent vs 28.6 percent, P=0.003). The most significant change due to bone base modification was an increase in recovery in the middle section of the cartilage. These results provide insight into mechanisms of cryoinjury in tissue systems.

Symmetry analysis of talus bone: A Geometric morphometric approach.

OBJECTIVE: The main object of this study was to use a geometric morphometric approach to quantify the left-right symmetry of talus bones. METHODS: Analysis was carried out using CT scan images of 11 pairs of intact tali. Two important geometric parameters, volume and surface area, were quantified for left and right talus bones. The geometric shape variations between the right and left talus bones were also measured using deviation analysis. Furthermore, location of asymmetry in the geometric shapes were identified. RESULTS: Numerical results showed that talus bones are bilaterally symmetrical in nature, and the difference between the surface area of the left and right talus bones was less than 7.5%. Similarly, the difference in the volume of both bones was less than 7.5%. Results of the three-dimensional (3D) deviation analyses demonstrated the mean deviation between left and right talus bones were in the range of -0.74 mm to 0.62 mm. It was observed that in eight of 11 subjects, the deviation in symmetry occurred in regions that are clinically less important during talus surgery. CONCLUSIONS: We conclude that left and right talus bones of intact human ankle joints show a strong degree of symmetry. The results of this study may have significance with respect to talus surgery, and in investigating traumatic talus injury where the geometric shape of the contralateral talus can be used as control. Cite this article: Bone Joint Res 2014;3:139-45.

Histological scoring of articular cartilage alone provides an incomplete picture of osteoarthritic disease progression.

PURPOSE: To ascertain whether molecular subcategories of disease progression exist within established histological grades of articular cartilage (AC). METHODS: Based on H&E and safranin-O staining of AC sections obtained from 18 knee arthroplasty surgeries, 30 samples ranging from Mankin Scoring System grade 1 through 5 were identified. Immunohistochemical (IHC) analysis for collagen type II and aggrecan was performed on serial sections of the paraffin-embedded AC samples. Six AC samples from each of the five Mankin Scoring System grades were examined. RESULTS: Significant IHC differences in collagen type II and aggrecan deposition were seen within AC samples from all five histological grades. The range of IHC differences in collagen type II and aggrecan increased with increasing histological grade. A change in the pattern of collagen type II deposition was observed in MG-3 AC that was consistent with a switch in collagen type II metabolism. CONCLUSIONS: IHC staining of collagen type II and aggrecan can identify differences within histological grades of AC that are consistent with the existence of molecular subcategories. These differences were detectable even within the lowest histological grades; therefore the use of IHC staining can further enhance and refine the scoring of AC deterioration in early osteoarthritis (OA). Furthermore, the changes seen in the deposition pattern for both aggrecan and collagen type II suggest that they could be used to monitor key molecular events in OA progression. These findings also underscore the need for the development of IHC scoring criteria.

Storage of porcine articular cartilage at high subzero temperatures.

OBJECTIVE: Transplantation of osteochondral allograft tissue can treat large joint defects but is limited by tissue availability, surgical timing, and infectious disease transmission. Fresh allografts perform the best but requirements for infectious disease testing delay the procedure with subsequent decrease in cell viability and function. Hypothermic storage at lower temperatures can extend tissue banking time without loss of cell viability and, therefore, increase the supply of allograft tissue. This study investigated the effects of different cryoprotectant solutions on intact AC at various subzero temperatures. DESIGN: 10 mm porcine osteochondral dowels were immersed for 30 minutes in various combinations of solutions [(XVIVO, propylene glycol (51% w/w), sucrose (46% w/w)] cooled to various subzero temperatures (-10, -15, and -20 degrees C), and held for 30 min. After warming, 70 mum slices were stained with membrane integrity dyes, viewed under fluorescence microscopy and cell recovery calculated relative to fresh controls. RESULTS: Results demonstrated excellent cell recovery (>75%) at -10 degrees C provided ice did not form. Excellent cell recovery (>70%) occurred at -15 degrees C in solutions containing 51% propylene glycol but formation of extra-matrix ice in other solutions resulted in significant cell loss. All groups had <6% cell recovery at -20 degrees C and propylene glycol did not provide a protective effect even though extra-matrix ice did not form CONCLUSIONS: These results suggest that extra-matrix ice plays an important role in cell damage during cryopreservation. Excellent cell recovery can be obtained after storage at subzero temperatures if ice does not form. Hypothermic preservation at high subzero temperatures may extend AC storage time in tissue banks compared to current techniques.

Intra-articular mechanical blocks and full extension in patients undergoing anterior cruciate ligament reconstruction.

Patients with acute anterior cruciate ligament (ACL) rupture frequently present with a lack of full extension. Current literature is unclear whether arthroscopic debridement is necessary before reconstruction to achieve full extension postoperatively. This study examined the postoperative extension achieved in 153 knees that underwent ACL reconstruction within 12 weeks of index injury. All patients performed preoperative physical therapy to increase range of motion and control pain/swelling, regardless of presenting range of motion without prior aspiration or arthroscopy. Of the 153 knees, 103 had meniscal pathology, of which 73 were peripheral vertical tears; 96 of the 153 knees lacked >/=3 degrees extension preoperatively. Five of 96 knees had an intra-articular mechanical block to extension and all regained full extension after ACL reconstruction. This study documented that a true intra-articular mechanical block is unusual in primary ACL ruptures. Lack of full extension can be adequately dealt with during surgical reconstruction without a detrimental effect on knee extension postoperatively.

Effect of varying angles on the pullout strength of interference screw fixation.

Arthroscopically assisted reconstruction of the anterior cruciate ligament-deficient knee using a bone-patellar tendon-bone graft is a new and evolving technique. One technically demanding aspect involves the placement under arthroscopic visualization of the femoral interference screw. The effect on pullout strength of changing the angle of the interference screw from 0 degree to 10 degrees, 20 degrees, or 30 degrees with respect to the bone plug was examined. The mean pullout strengths were 621 +/- 82, 594 +/- 48, 508 +/- 66, and 485 +/- 62 N, respectively. We concluded that there was no significant difference in the tensile strength provided by interference screw fixation for angles up to 10 degrees, but that there was a significant (p = 0.0010) weakening of fixation for screw angles > or = 20 degrees.

Ultrasonography in developmental hip dysplasia.

With the evolving use of ultrasonography, there has been an increasing emphasis on the technically oriented diagnosis of developmental hip dysplasia. Graf's alpha and beta angles and Harcke's bony rim percentage are two of the more common criteria, but their repeatability and reliability have not been well documented. Fifty-eight hip ultrasounds of 29 infants were measured independently by three physicians, and statistical analysis was performed. It was concluded that Graf's and Harcke's methods could be repeatable with training and frequent use of ultrasonography. Harcke's guidelines have a large equivocal zone, which limited their clinical usefulness. Graf's angles gave a better classification, but their clinical relevance has not been proven.

Long-term osteoarthritic changes in anterior cruciate ligament reconstructed knees.

To consolidate the indications for anterior cruciate ligament reconstruction and clarify the long-term prognosis associated with current surgical and rehabilitation techniques, the incidence of osteoarthritis in arthroscopically anterior cruciate ligament reconstructed knees requires investigation. Seventy-two patients with anterior cruciate ligament ruptures who were active in sports requiring sidestepping and pivoting, or who had recurrent episodes of giving way, underwent arthroscopic bone-patellar tendon-bone anterior cruciate ligament reconstruction. These patients were evaluated for meniscal damage and osteoarthritic changes at the time of surgery and followed up for 7 years. Fifty-three patients underwent radiographic evaluation at 7 years, which included anteroposterior, lateral, skyline, and 30 degrees posteroanterior weightbearing views. Radiographic evaluation was performed by three independent surgeons and graded as per International Knee Documentation Committee criteria. Results revealed that knees with chronic anterior cruciate ligament deficiency, even those with intact menisci before reconstruction, suffered early osteoarthritic changes. More severe changes were seen with meniscectomy. Acute anterior cruciate ligament reconstruction with meniscal preservation was shown to have the lowest incidence of degenerative change. Controversy exists regarding the timing of anterior cruciate ligament reconstruction. This study supports early reconstruction of anterior cruciate ligament deficient knees before episodes of giving way occur in individuals intent on continuing activities that involve sidestepping and pivoting.