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Keywords:

  • MACI;
  • confocal microscopy;
  • cell viability;
  • knee;
  • chondrocyte

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

Cell viability is crucial for effective cell-based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix-induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre-operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 105 cells/cm2 (5.74–7.11 × 105) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4–77.8%). This dropped to 67.4%(64.1–69.7%) after handling (p = 0.002), 56.3%(51.5–61.6%) after being thumbed (p < 0.001) and 28.8%(24.7–31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2–18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser-scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix-induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1495–1502, 2014.

Autologous chondrocyte implantation (ACI) was first reported by Brittberg et al. (1994) for the repair of full thickness articular cartilage defects of the knee.[1] In this technique, mature chondrocytes are released from an autologous cartilage biopsy, expanded ex vivo in culture and then re-inserted under a periosteal flap. Since then, ten-year results from a prospective randomized control trial have demonstrated improved results for ACI compared to mosaicplasty.[2]

ACI using periosteum as a cover for the cells can result in hypertrophy of the graft.[3] First generation ACI was also unable to address uncontained articular cartilage defects at the margin of joints effectively. Accordingly, alternative carriers, including type I/III collagen membranes, were introduced to mitigate these effects. This evolved into matrix-induced autologous chondrocyte implantation (MACI),[4] where the autologous cultured cells were seeded onto a collagen I/III membrane in the laboratory and delivered to the surgeon as a construct. Genzyme (Geel, Belgium) term their collagen membrane ACI-Maix™ a similar membrane is available without cells as Chondro-Gide® (Geilstlich Pharma AG, Wolhusen, Switzerland). It has a bilayer structure with a compact smooth surface that is impermeable to cells which prevents the cells diffusing into the synovial fluid. The other layer has a loose porous nature that favours cell invasion and attachment. This environment has been shown to encourage chondrocyte phenotype and production of collagen II and glycosaminoglycans.[5, 6] The membrane is cut to shape at the time of surgery and can be secured in place with Tisseel fibrin glue (Baxter, IL, US). Bartlett et al. (2005) found no difference in their prospective randomised study between ACI and MACI at one year.[7] The SUMMIT trial has demonstrated superior clinical results of MACI compared to microfracture at two years.[8] MACI is technically simpler and able to address uncontained defects at the margin of the knee, hence has gained popularity.

The reasons why cartilage repairs may fail to integrate with host cartilage, and therefore fail clinically, have been the subject of a recent review.[9] One key factor is that mechanical trauma causes chondrocyte death as there is a band of cell death when native cartilage is cut.[10] Gigante et al. (2007) used a colorimetric assay of MTT (dimethylthiazol-diphenyltetrazol bromide) and estimated the density of live cells seeded onto a collagen membrane to be 5–110 × 103 cells/cm2 despite an initial seeding density of 1 × 106 cells/cm2.[11] However, there was considerable variability in the density of cells on the membrane, the live cells were not individually identified and therefore the proportion of dead cells could not be evaluated.

Confocal laser scanning microscopy (CLSM) has been used to evaluate the viability of chondrocytes in articular cartilage using fluorescent indicators which label living or dead cells.[12-15] CLSM has also been used in vivo to evaluate the effect of MACI[16] but has not, to our knowledge, been used to visualise the cells on the membrane ex vivo. The effects of trauma[12] and resulting zones of chondrocyte death and mechanisms of chondroprotection[13] have been assessed in cartilage. We are not aware of any published data using CLSM to evaluate the density and viability of the chondrocytes seeded onto on a MACI membrane. Nor are we aware of any published data on the effects of the mechanical trauma that would be expected during surgery.

The primary aim of this study was to quantify the number of live and dead cells on the collagen membranes used for MACI, and to determine the effects of the MACI implantation, on chondrocyte viability and density. We hypothesised that confocal microscopy would permit the quantification of chondrocyte density, viability and morphology of individual cells on MACI membranes, and that the actions required to implant a MACI membrane would cause chondrocyte death.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

The cell-seeded membranes used for this study were obtained from five patients undergoing a MACI repair to the knee or ankle. All patients were under the age of 40 years old. Patients were consented prior to surgery for study of material that would normally be discarded. All samples were incinerated following analysis. Consultation with the local ethics committee confirmed that ethical approval was not required to study non-identifiable material in accordance with donor consent.

Cartilage was harvested arthroscopically from the minimally weight-bearing area of the ipsilateral knee. The biopsies were sent to Genzyme where the extracellular matrix was digested, the released cells were expanded in culture and then seeded onto a collagen I/III membrane using proprietary technology. The cells were expanded until there were approximately 1–2 × 107 cells available to seed onto a 4 × 5 cm collagen membrane (density approx. 0.5–1 × 106/cm2). A 1 × 5 cm section was removed by Genzyme (for quality control checks) and the remaining 3 × 5 cm piece was transferred to the surgeon for implantation. The quality checks include quantitative real-time polymerase chain reaction for identity (chondrogenic marker HAPLN1, synovial/fibroblastic marker MFAP5) and potency (aggrecan).[17] All of the matrices were delivered in the normal manner within 48 h of the cell seeding process being completed. There was a period of approximately six weeks from biopsy to implantation.

MACI membranes were subjected to several experimental protocols to represent various surgical stages throughout the MACI procedure. These groups along with the conditions that were applied, and what stage this represented during the MACI insertion are detailed in Table 1 and demonstrated in Figure 1. The first group represents the membrane as it is delivered to the surgeon. The second and third groups were obtained during surgery and the last two modeled the possible adverse effects of surgery ex vivo. For the final group[5] a piece of handled membrane was covered with a piece of paper with a hole in it and was deliberately crushed once using a pair of forceps to represent the effects of ‘heavy handling’. The hole allowed accurate identification of the area injured when it came to imaging.

Table 1. The Groups That Were Used in the Study Clarified by the Modeling They Underwent and What This Represents in the MACI Insertion
GroupExperimental Conditioning of the MembraneSurgical Stage Modeled
1Prior to handing during surgery.Delivery grade membrane.
2After handling to allow cutting out of membrane for repair.Minimal amount of handling required during surgery.
3Edge of the residual membrane adjacent to where a piece has been cut out.The edge of the edge of the inserted membrane.
4Duplicate piece of membrane cut out and thumb pressure applied ex vivo.Membrane on insertion and after being thumbed into place.
5Residual, handled membrane, crushed with forceps.Inadvertent ‘heavy’ handling.
image

Figure 1. Diagram demonstrating the samples taken and the experimental conditions applied.

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Images were taken from the middle of the membranes apart from specimens examining cut edges. Five images were obtained for each of the groups being analysed. Where possible, each image within a group was from a different patient. Due to the varying amount of membrane available this was not possible for all images. Membranes were prepared by one of the authors (LCB) and three other surgeons (see acknowledgements).

Samples were collected during or immediately after surgery and kept in their transport media until stained. All samples were incubated within 2 h of collection with live and dead cell fluorescent stains using 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI) respectively (Invitrogen, Paisley, UK). CMFDA stains the cytoplasm of live cells and the PI stains the nuclei of dead cells. The staining media was prepared by dissolving 100 µg CMFDA in dimethyl sulphoxide; this was then added to Dulbecco's Modified Eagle Medium (Gibco®, Edinburgh, The United Kingdom) with 20 µg PI. The final concentration of CMFDA and PI were 43 µM and 6 µM respectively. The membranes were incubated in the dark at 37°C and 5% CO2 for 45 min. The membranes were then carefully washed three times with phosphate buffered saline (PBS) and fixed in 10% formalin in the dark at RT for 30 min. After further washing with PBS, the membranes were stored in PBS in the dark at 4°C until analysis. All samples were imaged within 24 h of staining.

Imaging was performed using a Zeiss Axioskop LSM 510 (Carl Zeiss Ltd, Welwyn Garden City, UK) confocal laser-scanning microscope (CLSM). The x10 objective was used to determine the overall appearance and for cell counting to determine the density and viability of the chondrocytes. The x63 objective was used to determine the fine morphology of the chondrocytes. A multi-track protocol involving argon and helium–neon laser excitation, bandpass filters (500 nm to 550 nm) and long-pass filters (> 560 nm) allowed separation and measurement of the fluorescence emitted from CMFDA (λex = 488 nm, λem = 517 nm) and PI (λex = 543 nm, λem = 650 nm). Axial images were collected throughout the entire fluorescently visible z-axis at intervals of 5.7 µm for the ×10 objective and 0.75 µm for the x×63 objective.

Image analysis and three-dimensional reconstruction of the cells were performed with Velocity 6.1 software (Perkin Elmer Ltd., Massachusetts, USA). The same region of interest (900 × 900µm) was used for cell counting in all samples except for the group visualizing the cut edge (Fig. 2). The images with a cut edge were split into three sections: (i) No cells; (ii) Majority dead cells; (iii) Majority live cells. The method used to create these sections is demonstrated in Figure 2A, where lines were drawn between section one and two (line 1) and between sections two and three (line 2). The width between these two lines was used to represent the band of cell death. A third line was drawn the same distance into section three for comparison of the adjacent membrane (line 3).

image

Figure 2. Image of a cut edge of a MACI membrane visualised using the x10 objective. This represents the circumferential border of the construct to be inserted into the cartilage defect. The cut edge is at the bottom of the image in (A) and on the left in (B). (A) ×10 magnification demonstrating the lines used to measure the cut and adjacent regions of interest (B) 3D reconstruction of the same piece demonstrating deformation of the edge of the membrane. Scale bar=150µm.

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Quantification of live and dead cells were calculated using a cell counting protocol based on cell size and voxel intensity thresholding.[12, 13, 15, 18, 19] The protocol was set by a single investigator (PH) and kept constant between images; the cell count was then completely automated. These data were used to calculate the cell density (total number of cells/area) and percentage of cell death (number of live cells/total number of cells ×100). These data are presented as the mean and the range.

Data were analysed using multivariate analysis of variation (MANOVA). Levene's test for equality was used to determine homogeneity of variance. Significant values were subsequently followed up using one-way univariate analysis of variation (ANOVA). Tukey's post hoc test was used to determine significance between groups. All data analysis was performed using SPSS (IBM, version 21). A p-value <0.05 assumed significance.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

Chondrocytes were visualised on the membrane using x10 and ×63 objectives (Figs. 26). The collagen membrane does not stain with the fluorescent probes used in this study so only the cells were visible. Using these images we quantified the number of live and dead cells and observed their morphology.

image

Figure 3. Appearance of cells upon delivery imaged at ×10 magnification. (A) Extended focus view demonstrating distribution in the x–y plane (B) 3D reconstruction to illustrate the cell distribution in the z axis. Note that the membrane is not fluorescently labeled and therefore cannot be seen beneath the cells. Scale bar = 150µm.

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image

Figure 4. MACI membrane that had been handled during surgery and a sample collected prior to the repair piece being taken. (A) Magnification at ×10 demonstrating increased dead chondrocytes compared to Figure 2 (B) Magnification at ×63 showing cells without the typical rounded appearance of chondrocytes. Scale bars = 150µm and 20 µm respectively.

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image

Figure 5. Images from the middle of a membrane after being handled, cut into shape and then pressed with a thumb to duplicate the effect of being secured into the cartilage defect. Scale bar = 150µm.

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image

Figure 6. Membrane grasped with toothed forceps to simulate the effect of heavy or inadvertent handling (×10). There is a decreased cell density with a higher proportion of dead cells (see pooled data in Table 2). Scale bar = 150µm.

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The delivery grade membrane (Fig. 3A) showed a heterogeneous distribution of live and dead chondrocytes. There were relatively few nuclei from dead cells and they were not localised to a particular area. The 3D reconstruction that visualised the cells from the side (x-z axis) demonstrated a flat profile (Fig. 3B). Figures 4A and B demonstrate a sample that had been handled during surgery and imaged at x10 and x63 magnification respectively. The image taken at x10 magnification demonstrates increased cell death compared to the delivery grade seen in Figure 3. The higher magnification allowed visualisation of the cell morphology. The stained cytoplasm demonstrated cells that did not exhibit the spherical shape that would normally be expected from mature chondrocytes within native cartilage.[20] To ensure the membrane fits precisely during a MACI, the membrane is cut to shape during surgery. The images in Figure 2 were from the residual cut edge where the membrane was removed for the repair. There was a zone of cell death around the perimeter that was measured three times in each of the images with a cut edge; the mean width of the cut area was 275 µm (197–364 µm). The 3D reconstruction in Figure 2B is from a specimen with a cut edge. This demonstrates that the cells are not flat as they are when they are delivered (Fig. 3B). As the membrane structure is consistent this difference is probably due to cutting rather than variation in membrane structure.

Tisseel fibrin glue is used to attach the MACI membrane into the articular cartilage defect and the membrane is then secured into place using firm pressure, usually from the thumb. This pressure was duplicated and can be seen in Figure 5. Some areas demonstrated minimal difference to a piece that had not been thumbed whilst others demonstrated increased cell death.

A piece of the handled membrane was deliberately crushed between the tips of fine forceps. This area was labelled to allow accurate identification at the time of imaging. This piece is shown in Figure 6 and demonstrates increased cell death compared to the piece that was handled. Areas adjacent to this were visualised as controls and no difference was found compared to handled membrane.

All assumptions were met for the use of MANOVA for statistical analysis. Using Pillai's trace, there was a significant effect of mechanical trauma on the cell density and viability, V = 1.93, F(10, 48) = 131.9, p < 0.001. Levene's test demonstrated homogeneity of variance for both dependent variables (cell density p = 0.316, cell viability p = 0.519). Subsequent univariate ANOVAs revealed significant effects on both cell density, F(5, 24) = 132.8, p < 0.001, and cell viability, F(5, 24) = 379.4, p < 0.001. Tukey's post hoc analysis was used to determine significant differences between the groups.

Cell viabilities for each group are displayed in Table 2. Comparisons between delivery grade and handled membranes are shown as well as between the handled membrane and all the other groups. The data show that there was a significant decrease from 75.1% to 67.4% in the cell viability after the membrane has been handled (p = 0.002). As the membrane has to be handled during surgery, this was the specimen used as baseline for comparison to the other test conditions. There was a significant decrease from 67.4% to 56.3% after thumb pressure had been applied to the membrane (p < 0.001). The cell viability in the area where the handled membrane was cut decreased to 13.7% (p < 0.001), the area adjacent to this also significantly decreased to 61.3% (p = 0.023). Where the membranes had been crushed the viability dropped to 28.8% (p < 0.001). The cell viabilities are displayed in Figure 7.

Table 2. The Percentage of all Cells That Were Live are Presented as Mean (range)
Group 1Percentage Live Cells (%, range)Group 2Percentage Live Cells (%, range)p-value
  1. Differences between the conditions in groups 1 and 2 were analysed using Tukey's Post Hoc analysis.

Delivery75.1 (72.4–77.8)Handled67.4 (64.1–69.7)0.002
Handled67.4 (64.1–69.7)Thumbed56.3 (51.5–61.6)<0.001
  Adjacent61.3 (58.7–63.6)0.023
  Cut13.7 (10.2–18.2)<0.001
  Crushed28.8 (24.7–31.2)<0.001
image

Figure 7. Comparison of cell viability by group. Five images were analysed for each group and presented as mean with standard error bars. MANOVA with Tukey's post hoc analysis *= p < 0.05, **= p < 0.01, *** = p < 0.001.

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Cell densities for each group are displayed in Table 3. Comparisons between delivery grade and handled membranes are shown as well as between the handled membrane and all the other groups. The cell density (live and dead) was 6.60 × 105  cells/cm2 (range 5.74–7.11 × 105) for those membranes that had not received significant trauma (delivery, handled, thumbed, adjacent zone). There was no significant difference in cell density after the membranes had been handled (p = 0.324). There were significant decreases in cell density compared to the handled membranes when the membranes were subjected to cutting (p = 0.004) or crushing (p < 0.001) but not when thumbed into place (p = 0.5). Cell densities are displayed in Figure 8.

Table 3. Density of Cells Per cm2 Presented as Mean (range)
Group 1Density (x105/cm2, range)Group 2Density (cells x105/cm2, range)p-value
  1. Differences between the conditions in groups 1 and 2 were analysed using Tukey's Post Hoc analysis.

Delivery6.34 (6.02–6.21)Handled6.85 (6.70–7.11)0.324
Handled6.85 (6.70–7.11)Thumbed6.42 (5.74–6.39)0.500
  Adjacent6.81 (6.42–7.26)1.000
  Cut5.83 (4.91–6.48)0.004
  Crushed1.66 (1.32–2.03)<0.001
image

Figure 8. Density of the total number of cells on the membrane (live and dead). Five images were analysed for each group and presented as mean with standard error bars. MANOVA with Tukey's post hoc analysis ** = p < 0.01, *** = p < 0.001.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

The percentage of viable cells was 75.1% when the membrane was delivered to the surgeon; this is higher than the minimum 70% that is guaranteed by Genzyme. There was a greater proportion of dead cells as the membranes were subjected to the conditions they would undergo during surgery with the final viability averaging 56.3%. This would equate to an average of 3.61 × 105/cm2 live cells being implanted. The relevance of this figure remains uncertain as the optimal density of cells for cartilage repair has not yet been determined.

When the effect of ‘heavy handling’ was modelled there was a significant decrease in both the cell viability and density (Fig. 6). Our method to represent heavy handling was crude, but it effectively demonstrates what can happen if accidents or inadvertent grasping occur. It is likely that there will be a spectrum of cell death (such as the handled piece in Fig. 5) between the delivery grade membrane (Fig. 3A) and the crushed specimen (Fig. 6) during surgery.

The band of cell death associated with cutting was an average of 275 μm wide. As the membrane has to be cut to shape for insertion there will be a band of cell death around the edge of the implanted construct. It has previously been shown that there is an area of cell death when articular cartilage is cut with a scalpel.[10] As the defect that is to be repaired has to be debrided back to a stable rim to insert the MACI, it is likely that there will be a circumferential zone of cell death in the native cartilage. There will therefore be a band of cell death where lateral integration is supposed to occur and this may be a confounding factor for long-term integration. The clinical significance of the zone of cell death is unknown.

This study demonstrated that the cells are not in a monolayer on the MACI membrane and that the dead cells were found closer to the membrane. One possible explanation could be that the deeper cells are less able to obtain nutrition from the culture and transport media during the process of cell seeding and delivery. As these cells will be the furthest away from the defect when the membrane is fixed in situ the importance of this remains uncertain.

Our data demonstrate that the number of cells seeded onto the membrane were in the range expected by Genzyme (our study 0.66 × 106, Genzyme 0.5–1.0 × 106). This is greater than the density of 5–110 × 103/cm2 that was reported by Gigante et al. (2007) who used a colorimetric assay to quantify cell viability.[11] The density of cells decreased significantly when the membrane was subjected to trauma such as heavy grasping and cutting (Table 3). This suggests that whilst the cells are sensitive to mechanical trauma it takes a considerable force to displace them from the membrane.

The high magnification images demonstrated that the chondrocytes on the matrix appear more flattened and spindle-like than typical rounded chondrocytes found in mature articular cartilage (Fig. 4B).[21] Possible reasons for the cells not appearing as typical chondrocytes include time in culture, culture conditions and a lack of normal mechanical loading. Zheng et al.[22] used scanning and transmission electron microscopy to image the cells seeded on MACI membranes. They observed rounded cells more typical of chondrocytes. The reasons for this difference are unclear; a possible explanation could be the different preparation methods between the two techniques. Higher magnification of samples means that smaller areas are imaged, which might not account for variability in cell phenotype across the entire membrane. Despite the cells not having a typical chondrocyte appearance, Genzymes' quality checks[17] and Zheng et al.[22] data suggest that the cells express a chondrocytic phenotype.

Obtaining images of sufficient definition and clarity to allow cell counting and assessment of morphology meant that relatively small areas were assessed. The limitation of this is that the images obtained may not fully represent the entire membrane. The setting up of the automated counting protocol requires a degree of subjectivity that can produce inter-experiment variation unless the settings are constant. These settings were maintained throughout our analysis and so this is not considered a factor in our results. Whilst handling and crushing are subject to variation this is also true in the clinical setting. Our results demonstrated a spectrum of damage that we feel reflects the possible damage that can occur during surgery.

Another potential limitation of this study is the relatively short time the cells were incubated prior to analysis. Previous studies have demonstrated that insults can result in time-dependent loss of cell viability.[12] It is possible that cell viability would decrease further after insertion.

This study demonstrates that confocal microscopy can be used to image chondrocytes on a MACI membrane. It has shown that whilst a degree of cell death is inevitable with the insertion of a MACI membrane there was still a large population of viable cells delivered into the cartilage defect. Our data highlight that surgeons should avoid repetitive handling and accidental grasping of the cell seeded membrane being inserted with instruments to maximize the number of live cells to repair the defect.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

The authors thank Dr. Trudi Gillespie of the University of Edinburgh's IMPACT Facility for her help with the confocal microscopy. We would also like to thank Mr. Tim White, Mr. John Keating and Mr. John McKinley for their help in acquiring the membranes used for the study. We would also like to acknowledge funding from the Edinburgh Orthopaedic Endowment Fund.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

All of the authors contributed to the design of the study. PH undertook the data acquisition and analysis. All authors contributed to data interpretation and the drafting and approval of the final manuscript.

ROLE OF THE SOURCE FUNDING

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES

Funding from the Edinburgh Orthopaedic Endowment fund covered the consumables and access charges to the confocal microscope. They had no further role in design, data interpretation or writing or approval of the manuscript.

CONFLICTS OF INTEREST

LCB has received a fee for speaking at an educational program for Genzyme/Sanofi and has attended the Sanofi Regenerative Medicine Strategy board meeting in 2011. PH and ACH have no interests to declare.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. AUTHOR CONTRIBUTIONS
  8. ROLE OF THE SOURCE FUNDING
  9. REFERENCES
  • 1
    Brittberg M, Lindahl A, Nilsson A, et al. 1994. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889895.
  • 2
    Bentley G, Biant LC, Vijayan S, et al. 2012. Minimum ten-year results of a prospective randomised study of autologous chondrocyte implantation versus mosaicplasty for symptomatic articular cartilage lesions of the knee. J Bone Joint Surg (Br) 94:504509.
  • 3
    Minas T. 2001. Autologous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop Relat Res S349361.
  • 4
    Frenkel SR, Toolan B, Menche D, et al. 1997. Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair. J Bone Joint Surg (Br) 79:831836.
  • 5
    Ehlers EM, Fuss M, Rohwedel J, et al. 1999. Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge. Ann Anat 181:513518.
  • 6
    Fuss M, Ehlers EM, Russlies M, et al. 2000. Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded onto a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopy study. Ann Anat 182:303310.
  • 7
    Bartlett W, Skinner JA, Gooding CR, et al. 2005. Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg (Br) 87:640645.
  • 8
    Saris D, Price A, Widuchowski W, et al. 2014. Matrix-Applied Characterized Autologous Cultured Chondrocytes Versus Microfracture: Two-Year Follow-up of a Prospective Randomized Trial. Am J Sports Med [Epub ahead of print] 10.1177/0363546514528093
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    Khan IM, Gilbert SJ, Singhrao SK, et al. 2008. Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review. Eur Cell Mater 16:2639.
  • 10
    Hunziker EB, Quinn TM. 2003. Surgical removal of articular cartilage leads to loss of chondrocytes from cartilage bordering the wound edge. J Bone Joint Surg Am 85-A:8592.
  • 11
    Gigante A, Bevilacqua C, Ricevuto A, et al. 2007. Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery. Knee Surg Sports Traumatol Arthrosc 15:8892.
  • 12
    Amin AK, Huntley JS, Bush PG, et al. 2009. Chondrocyte death in mechanically injured articular cartilage–the influence of extracellular calcium. J Orthop Res 27:778784.
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    Amin AK, Huntley JS, Patton JT, et al. 2011. Hyperosmolarity protects chondrocytes from mechanical injury in human articular cartilage: an experimental report. J Bone Joint Surg (Br) 93:277284.
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    Amin AK, Huntley JS, Simpson AHRW, et al. 2009. Chondrocyte survival in articular cartilage: the influence of subchondral bone in a bovine model. J Bone Joint Surg (Br) 91:691699.
  • 15
    Amin AK, Huntley JS, Bush PG, et al. 2008. Osmolarity influences chondrocyte death in wounded articular cartilage. J Bone Joint Surg Am 90:15311542.
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    Jones CW, Willers C, Keogh A, et al. 2008. Matrix-induced autologous chondrocyte implantation in sheep: objective assessments including confocal arthroscopy. J Orthop Res 26:292303.
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    Jomha NM, Anoop PC, Elliott JA, et al. 2003. Validation and reproducibility of computerised cell-viability analysis of tissue slices. BMC Musculoskelet Disord 4:5.
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    Lin G, Bjornsson CS, Smith KL, et al. 2005. Automated image analysis methods for 3-D quantification of the neurovascular unit from multichannel confocal microscope images. Cytometry A 66:923.
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    Glowacki J, Trepman E, Folkman J. 1983. Cell shape and phenotypic expression in chondrocytes. Proc Soc Exp Biol Med 172:9398.
  • 21
    Bush PG, Hall AC. 2003. The volume and morphology of chondrocytes within non-degenerate and degenerate human articular cartilage. Osteoarthr Cartil 11:242251.
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    Zheng M-H, Willers C, Kirilak L, et al. 2007. Matrix-induced autologous chondrocyte implantation (MACI): biological and histological assessment. Tissue Eng 13:737746.