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Keywords:

  • cartilage;
  • osteoarthritis;
  • osteochondral autografting;
  • animal model;
  • mesenchymal stem cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES

Mesenchymal stem cells (MSC) are increasingly replacing chondrocytes in tissue engineering based research for treatment of osteochondral defects. The aim of this work was to determine whether repair of critical-size chronic osteochondral defects in an ovine model using MSC-seeded triphasic constructs would show results comparable to osteochondral autografting (OATS). Triphasic implants were engineered using a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase, and a collagen I hydrogel chondral phase. Autologous MSCs were used to seed the implants, with chondrogenic predifferentiation of the cells used in the cartilage phase. Osteochondral defects of 4.0 mm diameter were created bilaterally in ovine knees (n = 10). Six weeks later, half of the lesions were treated with OATS and half with triphasic constructs. The knees were dissected at 6 or 12 months. With the chosen study design we were not able to demonstrate significant differences between the histological scores of both groups. Subcategory analysis of O'Driscoll scores showed superior cartilage bonding in the 6-month triphasic group compared to the autograft group. The 12-month autograft group showed superior cartilage matrix morphology compared to the 12-month triphasic group. Macroscopic and biomechanical analysis showed no significant differences at 12 months. Autologous MSC-seeded triphasic implants showed comparable repair quality to osteochondral autografts in terms of histology and biomechanical testing. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1586–1599, 2010

Localized articular cartilage defects are frequently encountered by practicing orthopedic surgeons, but their proper treatment remains a controversial subject. It is widely held that cartilage lesions larger than a critical size result in an increased risk of progression to osteoarthritis (OA).1 Osteochondral autograft techniques such as the Osteochondral Autograft Transfer System (OATS) or mosaicplasty are used in clinical practice for repair of localized femoral condyle cartilage lesions and have shown good long-term results.2, 3 However, this method presents a number of limitations. For one, morbidity at the donor site limits the size of defects that can be repaired to an optimal maximum size of 1–4 cm2.4 Post-operative symptoms related to the donor site such as persistent pain have been reported by several authors.5–7 Donor site bleeding has been implicated as a cause of early hemarthrosis.4, 8 In addition, the surgical difficulty of shaping host tissue to fit the defect area is a limiting factor.9 Inadequate bonding of the graft cartilage to surrounding tissue is a common finding noted in second-look arthroscopy in clinical subjects10 as well as histological analysis in animal models.11, 12

Synthetic tissue-engineered multiphasic implants consisting of distinct cartilage and bone layers are an area of increasing research interest. This treatment method has the potential to restore the anatomical osteochondral junction without iatrogenic injury at donor sites and with the flexibility to treat larger lesions.13 Biphasic structures cultivated with autologous osteogenic and/or chondrogenic cells have shown promising results in vitro.14–16 The majority of in vivo studies investigating multiphasic scaffolds have been performed on small animal models.17–20 Kandel et al.21 investigated biphasic constructs in a sheep model showing in vivo results after 9 months. This study suggests that biphasic constructs may be suitable to repair joint defects. However, this study used autologous chondrocytes for the cartilage layer instead of bone marrow-derived MSCs. In addition, nearly all of this research is done on a model of acute cartilage injury in which generation of a defect is immediately followed by repair in a single surgery. To more accurately model the chronic cartilage damage seen in human subjects undergoing cartilage repair, use of a chronic injury model may be more appropriate.22 Based on superior in vivo results for MSC-seeded constructs in comparison to chondrocyte-seeded implants23 and due to the demonstrated osteogenic and chondrogenic potential of MSCs,15, 18, 24–28 we developed a new MSC-based triphasic construct. The objective of this study was to compare the clinically established OATS technique with triphasic MSC-seeded implants for treatment of chronic osteochondral defects. We hypothesized that repair of critical-size chronic osteochondral defects using MSC-seeded triphasic constructs would show cartilage properties comparable to osteochondral autografting as well as good integration of the osseous phase. To the best of the authors' knowledge, no prior studies showing 12-month results of MSC-based multiphasic cartilage repair in a chronic large animal defect model have been published.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES

A complete description of the methodology used in this study can be found in Appendix A.

Animals

Ten skeletally mature and healthy female merino sheep with an age of 2–2.5 years and an average weight of 65 kg (57–75 kg) were used for this study. The sheep were randomly divided into two groups with follow-ups of 6 and 12 months. The right hind knee of each sheep was treated with a triphasic scaffold and the left knee was treated with an osteochondral autograft. The animals were treated in accordance with applicable animal protection laws (Paragraph 8, Section 1) and authorization by the local legal representative was granted (TVV/07).

Bone Marrow Aspiration and Triphasic Implant Production

Prior to the first surgery, bone marrow aspirates were obtained from each sheep and MSCs were isolated and expanded for 4 weeks to passage one. Venous blood was also collected from each sheep and autologous EDTA plasma and serum were isolated. For the chondral phase, 4.0 × 105 MSCs/mL were mixed with a clinically approved collagen type I hydrogel (CaReS®, Arthro Kinetics Biotechnology, Krems, Austria) and cultured for 14 days in a chondrogenic medium (Chondrogenic Bullet Kit®, Lonza, Cambridge) with 10 ng/mL TGF-β3. Simultaneously, 1 × 106 MSCs were mixed with autologous EDTA plasma and 0.1 M CaCl2 and seeded onto resorbable β-TCP implants (CERASORB®, Curasan, Kleinostheim, Germany) with a diameter of 6 mm and length of 10 mm. The cylinders contained four parallel-oriented, vertical macropores with a diameter of 1 mm, resulting in a true porosity of 65%. The osseous constructs were then cultured for 14 days in autologous expansion medium (Fig. 1).

In Vitro Analysis of Constructs

The viability of the MSCs in both composites was determined following culture using fluorometric live/dead staining. Gene expression profiles of the chondral constructs were determined using RT-PCR with primers as shown in Table 1 of Appendix A. Scanning electron microscopy (SEM) was performed to confirm cell morphology and distribution on the constructs. Immunohistochemistry of cryosections of the chondral phase was performed for aggrecan and collagen type II (Fig. 1).

Table 1. O'Driscoll Histological Score
 Cell Morphology (0–4)Toluidine Blue Staining (0–3)Surface Regularity (0–3)Structural Integrity (0–2)Thickness (0–2)Bonding to Adjacent Cartilage (0–2)Hypocellularity (0–3)Chondrocyte Clustering (0–2)Freedom From Degenerative Changes (0–3)Total
  • a

    p < 0.05 compared with triphasic 6 months.

  • b

    p < 0.05 compared with triphasic 12 months.

  • c

    p < 0.05 compared 6 versus 12 months inside the triphasic group.

  • d

    p < 0.05 compared 6 versus 12 months inside the OATS group.

OATS 6 months (mean ± SD)2.8a ± 0.91.8 ± 0.41.0 ± 0.80.7d ± 0.40.9 ± 0.50.5a ± 0.42.4 ± 0.31.7 ± 0.31.4d ± 0.613.1 ± 2.6
Triphasic 6 months (mean ± SD)1.9 ± 0.31.5c ± 0.31.5 ± 0.80.8 ± 0.60.7 ± 0.41.5 ± 0.52.0 ± 0.42.0 ± 0.01.5 ± 0.313.5 ± 2.4
OATS 12 months (mean ± SD)2.8b ± 0.72.2 ± 0.21.7 ± 0.61.4 ± 0.41.1 ± 0.51.3 ± 0.92.7 ± 0.61.3b ± 0.02.1 ± 0.416.5 ± 2.6
Triphasic 12 months (mean ± SD)1.5 ± 0.72.3 ± 0.61.0 ± 1.01.1 ± 0.60.5 ± 0.51.5 ± 0.52.2 ± 0.91.8 ± 0.31.6 ± 0.413.5 ± 4.5
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Figure 1. Overview of triphasic implant production.

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Surgical Techniques

In the primary surgery, bilateral full-thickness defects with a diameter of 4 mm were created to 2 mm below the calcified layer in the medial femoral condyles of the sheep using a medial arthrotomy (Fig. 2A). After 6 weeks, the animals were returned for a second surgery with implantation of the constructs. The original arthrotomy was reopened and the initial defects (Fig. 2B) were cored out using a 6.4 mm harvesting drill to a depth of 12 mm. In the right knee, an MSC-seeded β-TCP cylinder was inserted (Fig. 2C). Autologous cancellous bone taken from the harvested cylinder was used to fill the 200 µm gap between the β-TCP cylinder and the surrounding native bone. The intermediate activated autologous plasma phase was applied and a seeded collagen I gel phase was affixed and fitted to the condyle surface (Fig. 2D). In the left knee, the patella was released medially and laterally subluxated to allow for harvest of an osteochondral cylinder of 6.6 mm in diameter and 12 mm depth. The cylinder was implanted using a press-fit technique. After 6 or 12 months, the hind knees were explanted for analysis of regeneration. Each sample was first visually graded by a specialist orthopedist using the Brittberg ICRS Visual Scale.29, 30 This scale assesses the degree of defect repair, integration to the border zone and macroscopic surface appearance with a rating of 0–4, where 4 represents the best appearance. After biomechanical analysis of the samples using a ball indentation test, the samples underwent plastination for preparation of histological slides. Toluidine blue31 and Levai–Laczko32, 33 stains were used. The slides were graded independently by three reviewers using the ICRS Visual Histological Scale,34 the O'Driscoll Scale35 and the semiquantitative Siebert score.36

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Figure 2. Implantation of triphasic construct. (A) Fresh defect immediately after creation. (B) Chronic defect at re-arthrotomy after 6 weeks. (C) Osseous phase with visible β-TCP cylinder. (D) Chondral phase after implantation.

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High-resolution micro-computed tomography (µCT) scans were made of all triphasic constructs (La Theta™ LCT-100, Aloka, Inc., Tokyo, Japan). The slice thickness was set to 0.1 mm. Measurements of the sinking distance of the β-TCP cylinders were then made using computer software (ImageJ, NIH, Bethesda, MD) by drawing a tangent to the subchondral bone surface and measuring perpedicular from this to the highest point on the β-TCP cylinder.

Statistics

For assessment of statistical significance, non-parametric Mann–Whitney U tests were done using SPSS (SPSS, Inc., Chicago, IL). Statistical significance was set at p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES

See Appendix B for cell viability and extracellular marker accumulation/expression results.

Assessment of Tissue Regeneration

Macroscopy

Visual evaluation according to the ICRS Brittberg score revealed significantly higher scoring in terms of “macroscopic appearance” (OATS 6 months: 2.6 ± 0.6, 12 months: 2.8 ± 1.1; triphasic 6 months: 1.4 ± 0.6, 12 months: 2.0 ± 0.7) and “degree of defect repair” (OATS 6 months: 4.0 ± 0.0, 12 months: 3.8 ± 0.5; triphasic 6 months: 3.2 ± 0.8, 12 months: 3.2 ± 1.3) for the OATS group after 6 months (Fig. 3A and B). At 12 months the OATS group showed higher values for both parameters without being statistically significant. Nearly no difference in terms of “bonding to adjacent cartilage” was observed macroscopically at 6 and 12 months.

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Figure 3. Macroscopic views after 6 months of OATS (A), triphasic (B), and repair tissue at donor site (C).

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Histological Analysis

ICRS Visual Histological Scale

We found no statistical differences in overall scores between the OATS and triphasic groups after 6 and 12 months using the ICRS scale (Figs. 4–6). Analyzing the subcategories individually, we observed significantly higher scores in the OATS group for “matrix composition,” “cell distribution,” and “cell population” after 12 months (Fig. 7). O'Driscoll score: No significant differences between the total scores of the two groups could be observed (Fig. 8 and Table 1). As with ICRS scoring, we found significant differences here in “cell morphology” with higher values for the OATS group after 6 and 12 months. “Bonding to adjacent cartilage” was significantly better for triphasic implants than autografts after 6 months but not after 12 months. Within the triphasic group, the repair tissue showed significantly higher “toluidine blue staining” after 12 months than after 6 months. Within the OATS group, better “structural integrity” for the cylinder was observed after 1 year compared to the results after 6 months. In addition, less degenerative changes were found after 12 months. Siebert Semiquantitative Score: Total scores and subcategory scores showed no significant differences between the two groups at either endpoint. Although “bonding to adjacent cartilage” was greater in the triphasic implants at 6 months (OATS: 0.7 ± 0.6; triphasic: 1.5 ± 0.5), this difference was not statistically significant (p = 0.071). For the triphasic group we observed higher scores (p < 0.01) for “subchondral reconstruction” after 1 year (3.2 ± 0.7) compared to 6 months (0.8 ± 1.0) (Figs. 4–8, Table 1).

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Figure 4. Histology of triphasic construct repair tissue (Levai–Laczko stain). (A) Sinking of the osseous construct with incomplete subchondral bone coverage (6-month specimen, 500 µm bar). (B) Complete subchondral bone coverage (12-month specimen, 500 µm bar). (C) Five times magnification of rectangle shown in (B) (100 µm bar).

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Figure 5. Histology of OATS specimens (Levai–Laczko stain). (A) Severe cyst formation was noted in this 6-month specimen (500 µm bar). Arrows indicate original osteochondral implant borders. (B) 12-month specimen with incomplete cartilage bonding (500 µm bar) and (C): 5× magnification of rectangle shown in (B) (100 µm bar).

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Figure 6. Histology of triphasic specimen (A). Magnification of the same triphasic (B) and OATS (C) histology (Levai–Laczko stain) at 5× showing the native/regenerated cartilage border. Native cartilage is seen on the left in (B) and (C).

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Figure 7. ICRS visual histological score by subcategories showing 2× standard error (2SE). Differences of statistical significance are marked with the * symbol.

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Figure 8. O'Driscoll score, group totals showing 2× standard error (2SE).

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Biomechanical Testing

Indentation forces showed no significant differences between the 6-month triphasic (0.09 ± 0.04 N) and OATS (0.15 ± 0.10 N) groups or between the 12-month triphasic (0.15 ± 0.02 N) and OATS (0.11 ± 0.05 N) groups (Fig. 9). One measurement from the 12-month triphasic group was not included in the analysis due to a technical error. Significantly (p < 0.047) softer repair tissue in comparison to native cartilage (0.16 ± 0.04 N) was measured for the 6-month triphasic group (0.09 ± 0.04 N). At 12 months, no statistically significant difference between triphasic regenerated cartilage and native cartilage (0.13 ± 0.03 N) was observed.

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Figure 9. Ball indentation test showing 2× standard error (2SE).

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Micro-CT

Measurements revealed that in total four triphasic implants were sunken more than 3 mm below the subchondral bone plate. The average distance between the tip of the β-TCP implant and the subchondral bone was 2.6 mm after 6 months and 2.4 mm after 12 months.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES

The concept of a triphasic scaffold arose from experimentation with MSC-seeded matrix-associated autologous chondrocyte implantation (MACI) methods for repair of osteochondral defects that showed frequent dislocation of the cartilage matrix and subchondral cavity formation. In response to this, an MSC-seeded osseous phase was added to provide subchondral stability and an intermediate activated plasma layer was used for fixation. In addition, an experimental study showed superior results with in vivo cartilage repair when prior in vitro chondrogenic differentiation was induced.37 Based on these findings, this study describes a novel triphasic construct for repair of deep osteochondral defects using a collagen I scaffold seeded with chondrogenically differentiated MSCs, an intermediate layer of autologous plasma and a porous, seeded β-TCP cylinder. This implant was compared to the clinically established OATS technique using a validated large animal model for degenerated critical-size cartilage defects.22 In contrast to our first hypothesis, the OATS group did appear to have superior cartilage matrix morphology. However, histological analysis showed generally similar results between the osteochondral autograft and triphasic groups, as no significant differences were observed in the total sum of any of the 3 histological scores used for analysis. The superior cartilage bonding to adjacent native tissue seen in the 6-month triphasic group was a notable finding given the well-known issue of incomplete cartilage bonding in osteochondral autografts.10–12 It further demonstrates the capacity of MSCs to differentiate into various tissues38 and the ability of MSCs to achieve good bonding to surrounding native cartilage.39 Additional longer-term studies are required here to chart the course of repair provided by these implants.

Our second hypothesis was that the autografts and β-TCP implants would both show good bone integration. For the β-TCP implants, we even noted significant resorption of the implant and ingrowth of trabecular bone. This supports the good biocompatibility of the implants, a concern presented by a previous study examining non-porous calcium phosphate scaffolds.40 On the other hand, sinking of the β-TCP cylinders more than 3 mm into the underlying bone was noted in 4 of the 10 samples with accompanying new formation of a subchondral bone layer above the cylinder, a phenomenon that was not observed in the autograft groups. MSCs have osteogenic potential, particularly in combination with osteosupportive material, such as hydroxyapatite/β-TCP.41 If this bony overgrowth were derived from the MSCs seeded onto the β-TCP implant, one would assume a more polydirectional growth pattern originating from the β-TCP. Instead of this, we observed overgrowth from the cylinder's rim. Thus the new bone formation does not appear to originate within the β-TCP implant, but rather appears to grow from the subchondral bone at both sides of the defect zone until the implant is covered. This is in accordance with a study performed by Petersen et al.,40 which showed sinking of an implant with accompanying bone overgrowth. The regeneration of the subchondral layer over time is in line with similar long-term studies.40, 42 In another study, Guo et al.43 published results of cartilage repair using MSC-seeded β-TCP scaffolds, but did not report any sinking or dislocation of the implants. One possible explanation is that a defect depth of 4 mm measured from the cartilage surface was used. Assuming a cartilage thickness of 1.5 mm and a further 1–2 mm for the calcified layer and subchondral bone plate,44 the implants might not have been as deep inside the cancellous bone, allowing for superior fixation. Given the relative softness of the cancellous femoral condyle in comparison to the rigid subchondral bone plate, one might assume that under weight-bearing, the rigid β-TCP implant is pushed deeper into the cancellous bone. Comparable mean sinking values after 6 and 12 months indicate that this process most probably occurs in the early post-implantation phase. Pulliainen et al.45 reported sinking of PLDLA scaffolds and attributed this to impact on the joint surface causing cracks in the subchondral bone. This effect might also be related to the implantation technique used for the triphasic implants, which may not have provided the same immediate retention force as the press-fit implantation of the OATS cylinders. Further studies in which post-operative weight-bearing can be limited are needed to confirm this and MRI imaging of gross joints with in situ implants could provide more precise, quantifiable information.

Some possible shortcomings of the OATS method were also seen in our research. In 7 of the 10 autografts, subchondral peri-implant cyst formation was seen in histological analysis (Fig. 5A). This did not always correlate with the surface appearance of the cartilage, and the functional significance of this finding is unclear. However, reports of this in other animal model studies of osteochondral autografts lead us to believe that this is not an isolated occurrence.46, 47 We also noticed severe degeneration at the donor sites with incomplete tissue filling and substantial bone exposure (Fig. 3C). Although the significance of donor site morbidity remains a controversial topic, the poor healing response noted in this study would seem to correlate well with previous findings of poor tissue quality and postoperative pain. Use of a more extensive arthrotomy including patellar luxation for the OATS method is one factor that requires discussion. After plug transfer, the medial retinaculum was reconstructed and patellar tracking was tested to ensure normal alignment. Harvesting the osteochondral plug from the medial trochlea is a commonly used method, but this requires patellar luxation. Although we cannot be certain that this did not influence our final outcome, we feel that the following points make this unlikely. First, the animals were clinically observed two times a day in the early postoperative period with regard to wound healing and effusion. We found no differences between both knees, indicating that no additional hematoma or bleeding was present in the OATS-treated knees. Furthermore, no postoperative patellar luxation occurred. At the time of harvest, a normal gait pattern was observed in all animals. Third, the defect on the medial condyle was in the load-bearing area influenced by vertical load. Even if instability or lateralization of the patella would have been present, an effect on plug healing would be unlikely. Another point to discuss in this regard is the potential for different load-bearing in the hind limbs. We did not measure the load-bearing, which is a limitation of the study design. However the defects were created and later treated in the same size and location on both sides. By operating on both knees at the same time, unloading of one leg was difficult to obtain for the animal, thus ensuring similar load-bearing of both knees. Further limitations of this study included the small sample size used, which was limited by experimental board approval. The data obtained from this study will allow for accurate power analysis in future studies and provide support for larger sample groups. We did observe a high level of variability in the quality of repair tissue, as can be seen in the standard deviations of histological scores. Further studies should also include control groups such as microfracture or untreated defects for comparison with more widespread treatment modalities. Additional endpoints such as in vivo radiographs or MRI imaging, gait analysis and ROM testing could lend clinical relevance to this treatment modality.

In general, these findings indicate that the MSC-seeded triphasic implants used here cannot be considered as a superior treatment option due to the observed sinking phenomenon of triphasic implants and high variability in the results of the repair tissue. However, good osseointegration of the MSC-seeded β-TCP implants and superior bonding of MSC-assisted cartilage repair were observed. Based on these results, further studies should investigate superior implant fixation into subchondral bone. More basic research regarding the integration of the chondral and osseous phases as well as animal studies with a larger sample size will also be necessary before clinical applications can be developed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES

The authors would like to thank Dr. P. Madaj-Sterba and G. Lemm for animal care. This work was supported by the formel.1 program of the Medical Faculty of Leipzig (55/2005, 97/2007), by the German Research Foundation (BA 1025/2-1), and by the German Ministry of Education and Research (BMBF Grant 0313836; BMBF, PtJ-Bio, 0313909). No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

APPENDIX A: MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES
Isolation and Cultivation of Ovine MSCs

Prior to the first surgery, bone marrow aspirates of 20–40 mL were obtained from the iliac crest of each sheep. Mononuclear cells were isolated from the heparinized aspirates (500 IU [3.125 µg/mL] per mL; Ratiopharm, Ulm, Germany) by Ficoll density gradient centrifugation (density 1.077 g/mL; Biochrom, Berlin, Germany) and plated at 2×104 cells/cm2 in tissue culture flasks with Dulbecco's modified Eagle's medium (DMEM; Gibco, Karlsruhe, Germany) supplemented with 10% autologous serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (both Biochrom), referred to below as autologous expansion medium. Cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% O2 to 5% CO2 balanced with N2 in a tri-gas incubator (Thermo Fisher Scientific, Dreieich, Germany). Medium was changed twice weekly. After 10–14 days, the cells were detached using trypsin/EDTA (0.25%/0.05 mM; Biochrom), passaged at 5,000 cells/cm2 and cultured to reach 80–90% confluence of passage one (P1) before collagen I gel and β-TCP-cylinder preparation.

Isolation of Autologous Serum

Venous blood (100 mL) was collected from each sheep by puncturing the veins of both ears using S-Monovette blood withdrawal systems containing coagulation activator (Sarstedt, Nümbrecht, Germany). The monovettes were centrifuged for 10 min at 2,500g and 20°C. The serum phase was harvested and inactivated by heat (56°C, 30 min).

Isolation of Autologous Plasma

For autogenic plasma and fibrin glue production, 18 mL of venous blood was obtained aseptically from each animal using S-monovettes containing EDTA (Sarstedt). The collected EDTA/whole blood mixture was processed to plasma by centrifuging at 2,500g for 10 min at 20°C and removing the plasma fraction. The prepared plasma was transferred into 2 mL cryo tubes and stored at −80°C until seeding of the β-TCP cylinder with MSCs and the implantation procedure.

Implant Preparation (Fig. 1)
Preparation and Cultivation of the Chondral Phase

For preparation of the chondral phase, 4.0 × 105 MSCs/mL were mixed with a clinically approved collagen type I hydrogel (CaReS®, Arthro Kinetics Biotechnology) according to the manufacturer's instructions. For each construct, an aliquot of 1.8 mL cell–gel-suspension were transferred to 12-well plates (BD Falcon, Heidelberg, Germany) and cultured 14 days with serum-free, chondrogenic medium (Chondrogenic Differentiation BulletKit®) supplemented with 10 ng/mL TGF-β3 (both Lonza, Wuppertal, Germany). The differentiation was performed at 20% pO2 (Thermo Fisher Scientific). The chondrogenic medium was changed twice weekly (Fig. 1).

Preparation and Cultivation of the Osseous Phase

Resorbable pure-phase β-TCP CERASORB® cylinders with a diameter of 6 mm and length of 10 mm were supplied as a gift from Curasan. The cylinders were seeded with 1 × 106 MSCs of P1 and cultured for 14 days in autologous expansion medium. Per cylinder, 1 × 106 cells were mixed in 100 µL thawed autologous plasma and 1 µL of 0.8 M CaCl2 was added to achieve fibrin polymerization with a clotting reaction. The β-TCP ceramic cylinders were then soaked in a 96-well plate with this mixture, transferred into separate wells of a 24-well plate filled with 1 mL of autologous expansion medium (see above), and incubated at 37°C, 95% relative humidity in an air/5% CO2 atmosphere (Thermo Fisher Scientific). Medium was changed twice weekly. Each culture condition was carried out in triplicate.

Determination of Cell Viability

The viability of the MSCs in both composites was determined following culture by a fluorometric live/dead staining method (Mobitec, Goettingen, Germany), which allowed for quantification of the cellular uptake of calcein AM and ethidiumhomodimer. After taking out both scaffolds from the well plates, the constructs were washed with 5 mL PBS for 2 h each and afterwards incubated with fluorometric assay components for 30 min. The excitation and detection wavelengths were 515 and 635 nm respectively.

Total RNA Extraction, cDNA Synthesis, and RT-PCR

Gene expression profiles of chondral constructs were evaluated by using RT-PCR in a Primus 96 Plus PCR machine (MWG Biotech, Ebersberg, Germany). Two to three gels were digested with 2 mg/mL collagenase A solution (Roche, Basel, Switzerland) for 2 h at 37°C and resuspended in RLT lysis buffer (Qiagen, Hilden, Germany). Total RNA was purified using the RNeasy Mini Kit (Qiagen). Digestion of DNA was performed by applying 2 U RQ1-DNAse (Promega, Mannheim, Germany) at 37°C for 30 min. RNA concentration and purity were measured with a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) at 260 and 280 nm. Single-strand cDNA copies were generated from 1 µg samples of total purified RNA by using random primers and M-MLV reverse transcriptase (both Promega) according to the manufacturer's protocol.

The prepared MasterMIX contained 10× PCR buffer, 25 mM MgCl2, 0.5 U Taq polymerase (Qiagen), 8 mm dNTPmix (Fermentas, St. Leon-Rot, Germany) and 10 mm of sequence specific primers (Table A1; Operon Biotechnologies, Cologne, Germany). PCR reactions condition were as follows: 95°C for 5 min/95°C for 30 s, annealing for 30 s, 72°C for 30 s, and final extension at 72°C for 5 min. All samples were normalized with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Table A1. Primer Used for RT-PCR
GenesPrimer SequencesFrom SpeciesFragment Size (bp)Annealing Temperature (°C)Cycle Number
Aggrecan5′-ACGCCATCTGCTACACAGG-3′Ovis aries2195630
 5′-AAAGGCTCCTCAGGTTCTGG-3′    
Chondroadherin5′-GATACCTGGAGACGCTCTGG-3′Bos taurus3375630
 5′-GGCTTTCTTGGACCTCTTGG-3′    
Collagen 1A15′-CCAGTCACCTGCGTACAGAACG-3′Ovis aries2456728
 5′-GCCAGTGTCTCCTTTGGGTCC-3′    
Collagen 2A1 transcription variant 15′-CAGGATGTCCAGGAGGCTGG-3′Homo sapiens2896230
 5′-CCCAGGAGGTCCTTTGGGTC-3′    
Collagen 2A1 transcription variant 25′-CCAGGATGTCCGGCAACCAGG-3′Homo sapiens3536230
 5′-GGAGGTCCTCGAGGTCCCATG-3′    
GAPDH5′-CCACTGGGGTCTTCACTACC-3′Ovis aries3356230
 5′-AAGCAGGGATGATGTTCTGG-3′    
Link protein5′-TCAGGAACTACGGGTTTTGG-3′Bos taurus2606232
 5′-TTGGCCTAGAGATGGGGTAG-3′    
Sox95′-AGGTGCTCAAAGGCTACGAC-3′Homo sapiens2946228
 5′-GCGGCTGGTACTTGTAATCC-3′    
Scanning Electron Microscopy

Constructs for scanning electron microscopy (SEM) were rinsed with Sorenson's buffer (pH 7.2) and immersed and fixed at 4°C in 2% glutaraldehyde prepared in Sorenson's buffer for 2 h. Scaffolds were rinsed in Sorenson's buffer and postfixed in 1% osmium tetroxide (Plano, Marburg, Germany) for 2 h. Scaffolds were rinsed again and dehydrated in acetone series, dried in Critical Point Dryer CPD30 (Baltec, Tucson, AZ) sputter coated with platinum and examined in a Hitachi S4500 scanning electron microscope (Finchampstead, UK).

Immunohistochemistry

Immunohistochemistry of cryosections was performed using the two-step indirect method. The samples were embedded in Tissue Tek® (Sakura, Zoeterwoude, the Netherlands) sectioned at a thickness of 8 µm by a Leica CM3050S cryotome (Leica Microsystems, Nussloch, Deutschland). The sections were fixed for 5 min in acetone, blocked for 30 min (sheep serum 1:10 diluted in PBS) and incubated with the primary antibody (collagen type II: mouse monoclonal antibody [Clone: II-4C11; MP Biomedicals, Solon, OH], diluted 1:2,000 in PBS; aggrecan: monoclonal mouse antibody [Acris Antibodies, Herford, Germany], diluted 1:50 in PBS). After washing with PBS, the secondary antibody of peroxidase-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch, Cambridgeshire, UK; diluted 1:50 in PBS) was added for 1 h at 37°C. Immunostaining was developed by 3-amino-9-ethyl-carbazol substrate. Cell nuclei were counterstained with Meyer's hematoxylin (Lillie's Modification; DakoCytomation, Hamburg, Germany).

Surgical Techniques
Defect Application

Following bone marrow aspiration and blood draw, an initial operation was performed to create bilateral full-thickness osteochondral defects in the medial femoral condyles of the sheep. Preoperative sedation, orotracheal intubation, and anesthesia were performed under supervision of a veterinarian. Arthrotomy of the hind knee joints was performed using a medial arthrotomy under standard sterile conditions. The defects were created with a diameter of 4 mm and a depth of 2 mm below the calcified layer using a custom depth-limited drill (Aesculap, Tuttlingen, Germany). The sheep were then returned to the herd for degeneration of the defects. Regular post-operative wound checks were performed.

Triphasic Construct Implantation

After 6 weeks, the animals were returned to the laboratory for implantation of the constructs. The primary arthrotomy was reopened in standard fashion to expose the chronic lesions, all of which were noted to be grade 4 on the Brittberg ICRS Visual Scale. The initial defect was cored out using a 6.4 mm harvesting drill to a depth of 12 mm and the defect cylinder was removed using a graft harvester (Zimmer, Winterthur, Switzerland). The MSC-seeded β-TCP cylinder was inserted using a press-fit technique. The intermediate autologous plasma phase was then activated using CaCl2 and applied to the surface of the cylinder. The seeded collagen I hydrogel phase was affixed onto the plasma layer and fitted to the condyle surface using an impactor (Zimmer). The joint capsule and wound were then closed in standard layer-by-layer fashion.

Osteochondral Autograft Transplantation

Surgery continued on the contralateral side and the defect was removed with a coring drill as previously described. The patella was released medially and laterally subluxated using an extended parapatellar incision. A 6.6 mm diameter and 12 mm deep osteochondral cylinder was then harvested from the medial facet of the trochlea. The cylinder was implanted with a press-fit technique into the condylar defect crater using SDS instruments. After plug transfer, the medial retinaculum was reconstructed using Vicryl 2-0 sutures and patellar tracking was tested to ensure normal alignment.

Explantation

The hind knees were then dissected at 6 or 12 months for analysis of regeneration. A diamond band saw (E310, Exakt Apparatebau GmbH, Norderstedt, Germany) was used to cut blocks containing each defect region of approximately 1 cm in diameter and 3 cm in depth from the femurs of the sheep. The blocks were cut perpendicular to the articular surface. All samples were initially placed in PBS prior to biomechanical testing and transferred to formaldehyde at 24 h post-explantation.

Macroscopic Analysis

Prior to further evaluation, each sample was visually graded by a specialist orthopedic surgeon according to the Brittberg ICRS Visual Scale (Table A2).29, 30 This system assesses (I) degree of defect repair (defect filling), (II) integration to border zone and (III) macroscopic surface appearance.

Table A2. ICRS Visual Scale29, 30
 Points
I: Degree of defect repair
 In level with surrounding cartilage4
 75% repair of defect depth3
 50% repair of defect depth2
 25% repair of defect depth1
 0% repair of defect depth0
II: Integration to border zone
 Complete integration with surrounding cartilage4
 Demarcating border <1 mm3
 3/4th of graft integrated, 1/4th with a notable border >1 mm width2
 1/2 of graft integrated with surrounding cartilage, 1/2 with a notable border >1 mm1
 From no contact to 1/4th of graft integrated with surrounding cartilage0
III: Macroscopic appearance
 Intact smooth surface4
 Fibrillated surface3
 Small, scattered fissures, or cracks2
 Several, small, or few but large fissures1
 Total degeneration of grafted area0
Overall repair assessment (max. 12 points)
 Grade I: normal12
 Grade II: nearly normal11–8
 Grade III: abnormal7–4
 Grade IV: severely abnormal3–1
Histological Analysis

The samples were dehydrated using progressive alcohol concentrations and placed into light-curing embedding resin (Technovit 7200 VLC, Heraeus Kulzer, Hanau, Germany) for 2 days. Following this, infiltration with embedding resin was performed for an additional 13 days with agitation under vacuum conditions. The samples were then placed into molds, which were filled with embedding resin. Polymerization was performed in two steps using a commercial polymerization device (Histolux, Exakt Apparatebau GmbH). Treatment with low light intensity was followed by a second stage of high light intensity to complete the polymerization process over a total time of 10 h. The samples were kept overnight in an incubator (Memmert, Schwabach, Germany) before being cut and mounted onto slides.

The slides were then treated with toluidine blue31 and Levai–Laczko32, 33 stains and finally graded independently by three reviewers including a specialist orthopedic surgeon and a musculoskeletal pathologist using the ICRS Visual Histological Visual Scale (Table A3),34 the O'Driscoll Scale (Table A4)35 and the semiquantitative Siebert score (Table A5).36

Table A3. ICRS Visual Histological Scale34
 Points
I: Surface
 Smooth/continuous3
 Discontinuities/irregularities0
II: Matrix
 Hyaline3
 Mixture: hyaline/fibrocartilage2
 Fibrocartilage1
 Fibrous tissue0
III: Cell distribution
 Columnar3
 Mixed/columnar-clusters2
 Clusters1
 Individual cells/disorganized0
IV: Cell population viability
 Predominantly viable3
 Partially viable1
 <10% viable0
V: Subchondral bone
 Normal3
 Increased remodeling2
 Bone necrosis/granulation tissue1
 Detached/fracture/callus at base0
VI: Cartilage mineralization (calcified cartilage)
 Normal3
 Abnormal/inappropriate location0
Sum of points (max. 18 points)
Table A4. O'Driscoll Score35
 Points
I: Nature of predominant tissue
 (A) Cellular morphology
  Hyaline articular cartilage4
  Incompletely differentiated mesenchyme2
  Fibrous tissue or bone0
 (B) Safranin-O staining of the matrix
  Normal or nearly normal3
  Moderate2
  Slight1
  None0
II: Structural characteristics
 (A) Surface regularity
  Smooth and intact3
  Superficial horizontal lamination2
  Fissures: 25–100% of the thickness1
  Severe disruption, including fibrillation0
 (B) Structural integrity
  Normal2
  Slight disruption, including cysts1
  Severe disintegration0
 (C) Thickness
  100% of normal adjacent cartilage2
  50–100% of normal cartilage1
  0–50% of normal cartilage0
 (D) Bonding to the adjacent cartilage
  Bonded at both ends of graft2
  Bonded at one end, or partially at both ends1
  Not bonded0
III: Freedom from cellular changes of degeneration
 (A) Hypocellularity
  Normal cellularity3
  Slight hypocellularity2
  Moderate hypocellularity1
  Severe hypocellularity0
 (B) Chondrocyte clustering
  No clusters2
  <25% of the cells1
  25–100% of the cells0
IV: Freedom from degenerative changes in adjacent cartilage
 Normal cellularity, no clusters, normal staining3
 Normal cellularity, mild clusters, moderate staining2
 Mild or moderate hypocellularity, slight staining1
 Severe hypocellularity, poor or no staining0
Sum of points (max. 24 points)
Table A5. Semiquantitative Siebert Score36
 Points
(A) Surface characteristics
 Smooth2
 Somewhat irregular1
 Irregular0
(B) Cartilage thickness
 76–100% of the surrounding cartilage4
 51–75%3
 26–50%2
 <25%1
 0% (defect)0
(C) Bonding
 Apposition of both contact surfaces2
 One surface1
 No bonding0
(D) Subchondral reconstruction
 Even with surrounding bone4
 Slightly offset (25%)3
 Moderately offset (50%)2
 Significantly offset (75%)1
 No reconstruction/formation0
(E) Cartilage gap
 No gap4
 Gap up to 2×5% of the transplanted cartilage thickness3
 26–50%2
 51–75%1
 76–100%0
Sum of points (max. 16 points)
Biomechanical Testing

Prior to histological analysis, the cut sample cylinders underwent biomechanical analysis with a ball indentation test. An area near the center of the defect was used for testing to avoid a boundary effect. In addition, samples of native cartilage from the lateral condyle of the right femur of each animal were tested for comparison. The samples were held uniaxially in a ring clamp. To obtain near-physiological conditions, the clamp was mounted in a transparent PMMA tank filled with PBS at a pH of 7.4. The tank was mounted onto a two-dimensional miniature electromechanical goniometer stage, which was in turn mounted onto a linear X, Y stage. This setup allowed for a maximum load of 40 N. To evaluate the mechanical properties of the samples, a ball indentation test was used with a ISO 14577-2 compatible setup. A steel ball (X105CrMo17) with a diameter of 1 mm and a Young's modulus of 215 × 103 N/mm2 was used. A displacement-controlled regime was performed with a loading/unloading velocity of 10 µm/s and a maximum depth indentation of 200 µm. The indentation depths of 200 µm were determined in a preliminary test on untreated native knee joins as the optimum between substrate influence from the bone tissue and the contact problem.

Pre-testing contact force was 2 mN. During the way-controlled experiment the force and travel distance were measured independently by a load cell and a inductive displacement sensor.

In relation to the relative varied morphology and thickness of different tissues layer the measured force values were influenced by these and represent a mixed/averaged value.

APPENDIX B

Viability, Distribution, and Morphology of Embedded Cells

Primary ovine MSCs from P1 were mixed with both the collagen I hydrogel for the chondral phase and the autologous plasma for seeding onto the β-TCP-cylinder for the osseous phase. Both constructs were cultivated for 2 weeks, combined with autologous plasma and later implanted into the osteochondral defect site. Overall cell viability, distribution and morphology were determined. As represented in Figure B1A and B, both types of scaffolds showed a high cell viability of ∼95% after 2 weeks of 3D cultivation. Fluorescence microscopy of the MSC collagen gels (Fig. B1A) demonstrated a high density of living MSCs and few dead cells within the gel. Fluorescence staining with a live/dead kit was also performed 14 days after seeding and cultivation of the MSC-plasma suspension onto β-TCP cylinders. This showed similar results to microscopy with a homogeneous distribution of living MSCs and few dead cells. Cross-sectional imaging of both MSC-seeded matrices by SEM analysis prior to implantation showed the characteristic microstructure of both biomaterials, the compact collagen fibers (Fig. B1C) and the fibrin fibrillar structure (Fig. B1D). Both images illustrate the integration of MSCs into the respective framework with a typical dense cell occurrence in the peripheral zones. Horizontally oriented cell layers of MSCs with fibroblastic morphology were seen covering both biomaterials.

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Figure B1. Viability, distribution, morphology of MSCs in the chondral (A and C) and osseous (B and D) phases after 14 days in vitro. Fluorescence images of live (green) and dead (red) MSCs in the respective scaffolds as determined by fluorometric viability assay (A and B 1,000 µm bar). Scanning electron microscopy images showing typical cell distribution and morphology of MSCs (white arrows) in the chondral and osseous material (black arrows) (C and D 30 µm bar).

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Accumulation and Expression of Cartilage-Specific Extracellular Matrix Markers
RT-PCR

Chondrogenic differentiation of the cartilage phase was analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) (Fig. B2) and immunohistochemistry (Fig. B3). In the monolayer culture (Fig. B2, lane 1) there was no significant expression of the analyzed genes, except for weak expression of the early, immature splice variant of collagen 2A1, the transcription variant 1. Collagen 1A1, which is expressed constitutively by MSCs, was found at constant levels during 3D cultivation. In contrast, at the time of implantation (day 14), the MSC gels were demonstrated to express mRNA of the genes Sox9, aggrecan, chondroadherin, and link protein, which represent important markers of chondrogenesis (Fig. B2, lane 4).

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Figure B2. Time course of gene expression of Sox9, aggrecan, collagen 2A1 (transcription variants 1 and 2), chondroadherin and link protein in collagen-I-hydrogel cultures measured by RT-PCR. Expression levels were normalized to the housekeeping gene GAPDH. Lane 1: monolayer culture of P1; lanes 2–4: gel cultures of days 1, 7, and 14.

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thumbnail image

Figure B3. Immunohistochemical detection of aggrecan (A and B) and collagen type II (C and D) in collagen-I hydrogel cultures (50×, 200 µm bar). One day after gel preparation no aggrecan staining was visible (A). Fourteen days after chondrogenic differentiation strongly positive staining of aggrecan through the entire construct was evident (B). Similar to this result, no collagen type II was evident at day 1 (C) but was observed at day 14 (D).

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The constructs also showed expression of the mature splice variant of collagen 2A1, transcription variant 2, which is known to be expressed in a later stage of chondrogenesis and which is encoded for the major solid component matrix protein of hyaline cartilage. Thus, these results suggest progression of chondrogenic differentiation.

Immunohistochemistry

Chondrogenesis of MSCs at the protein level was examined by immunostaining for proteoglycan aggrecan and hyaline cartilage specific collagen type II after gel preparation (Fig. B3A and B) and prior to implantation (Fig. B3C and D). After 1 day the cryosections of the gels showed no evidence of these major cartilage matrix markers. In contrast, the accumulation of aggrecan and collagen type II were evident after 14 days in the MSC hydrogels cultured under chondrogenic conditions, as demonstrated by the strong positive immunostaining. The immunohistochemical results showed a pronounced, strongly positive interterritorial staining of aggrecan throughout the entire construct, whereas collagen type II showed a distinct but not uniform distribution of staining within the hydrogels. These findings at the protein level in the MSC gels provide additional evidence of chondrogesis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. APPENDIX A: MATERIALS AND METHODS
  8. REFERENCES
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