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Stabilized Autologous Fibrin-Chondrocyte Constructs for Martin Fussenegger, MD,* Johann Meinhart, PhD,† Walter Höbling, MD,‡ Werner Kullich, PhD,§ Siegfried Funk, MD,¶ and Gu¨nther Bernatzky, PhD࿣ method, because it is able to overcome many of the problems Abstract: Stabilization of fibrin-chondrocyte constructs with fi-
brinolytical inhibitors has been shown to be a feasible method for
associated with traditional cartilage replacement methods.2 In the reconstruction of cartilage in vitro. In this study, the method was several applications, tissue engineering has already made a tested in vivo. Autologous cultures were used to form stabilized successful transition from a scientific method to a clinical fibrin-chondrocyte constructs that were injected into auricular car- procedure.3–6 The successful reconstruction of a phalanx by tilage defects of rabbits. Stabilization was achieved by high doses of bone tissue engineering in a human has recently been report- fibrinolytic inhibitors. Samples were prepared for magnetic reso- ed.7 Successful tissue engineering is dependent on numerous nance imaging, histology, and immunohistochemistry after 1, 2, 4, factors, but adequate scaffolds are among the most important and 6 months. Defects of the contralateral ear, which were treated prerequisites for stable three-dimensional and histiotypic tis- with stabilized fibrin without cells, were used for controlled com- sue. Numerous synthetic and natural polymers have been parisons. In all cell-fibrin samples, cartilage-like tissue was present.
tested as scaffolds for tissue engineering.8–12 One natural Immunohistochemistry revealed the presence of collagen II. Thisfinding was similar for all observations. In the control samples, only polymer is fibrin, which is increasingly used for tissue engi- minor new cartilage could be detected at the cut edges. The recon- neering. It can easily be polymerized and molded from its struction of cartilage in vivo by injecting fibrin-chondrocyte con- basic constituents. It is noncytotoxic, biocompatible, and structs, stabilized with inhibitors of fibrinolysis, is thus possible.
biodegradable and has been in clinical use for several years.
However, fibrin is unstable and is quickly disintegrated by (Ann Plast Surg 2003;51: 493– 498) cells. An in vitro study demonstrated that fibrin is an adequatescaffold for the reconstruction of cartilaginous tissue whenstabilized by antifibrinolytic substances.13 These fibrin-chon- Cartilage has a very slow turnover at the cellular and drocyte constructs were stable for 4 weeks in vitro. The cells molecular levels and therefore has a limited capacity for appeared to be viable and produced an extracellular matrix self-repair. Small, noncritical defects can regenerate in a low typical for cartilage. The present study tested the potential of percentage of cases, but full-thickness defects of critical size stabilized fibrin-chondrocyte for cartilage reconstruction in are replaced by tissue of inferior quality.1 Classic methods of an animal model. Auricular cartilage defects in rabbits were cartilage replacement such as autografting, allografting, and created and treated with stabilized fibrin-chondrocyte con- the use of synthetic materials are not ideal. Tissue engineer- structs. Neocartilaginous tissue was present in all treated ing was therefore proposed as a cartilage replacement defects. No to only minor neocartilaginous tissue was foundin sham-treated control defects.
Received August 7, 2002, and in revised form February 3, 2003. Accepted From the *Department of ENT, Head and Neck Surgery, General Hospital, MATERIALS AND METHODS
Wels, Austria; †First Department of Surgery, Lainz Hospital, Wels,Austria; ‡First Department of Pathology, General Hospital, Wels, Aus- Experimental Design
tria; §Ludwig Boltzmann Institute for Rehabilitation, Saalfelden, Austria; Twelve animals were treated with injectable stabilized ¶Second Department of Radiology, General Hospital, Wels, Austria; andthe ࿣Central Animal Facility, University of Salzburg, Salzburg, Austria.
Supported by Bauer Optics Ltd., Wels, Upper Austria.
Reprints: Johann Meinhart, PhD, Department of Surgery, Lainz Hospital, Wolkersbergenstr.1, A-1130 Vienna, Austria. [email protected].
Surgical procedure 1: Excision of two cartilage seg- Copyright 2003 by Lippincott Williams & Wilkins ments from one ear of each animal, one with and one without 0148-7043/03/5104-0493DOI: 10.1097/01.sap.0000067726.32731.E1 Annals of Plastic Surgery • Volume 51, Number 4, October 2003 Annals of Plastic Surgery • Volume 51, Number 4, October 2003 anced salt solution (Gibco, Paisley, UK) and incubated for 1 Control defects: The defects created during surgical hour in a 0.05% trypsin solution (Gibco). The tissue was then procedure 1 were filled with stabilized fibrin gel and served as mechanically sliced into pieces approximately 1 mm2 in size.
These were subsequently incubated in a solution of 0.1%collagenase CLS 2 (Worthington Biochemical Corp., Free- hold, NJ) in phosphate buffered saline without Caϩϩ and Cell culture: From the excised segments without the Mgϩϩ (Gibco), shaken in Erlenmeyer tubes (Corning Glass perichondrium chondrocyte cell cultures were established.
Works, Corning, NY), for 24 hours at 37°C in a shakingwater bath.
After incubation, the suspension was filtered through a Surgical procedure 2: After 12 days, these cultures 100-␮m nylon cell strainer (Falcon, Franklin Lakes, NJ) and could be used for treatment. Therefore, a second operation centrifuged. The pellet was resuspended in Medium-199 had to be performed. Two new defects were created in each (Gibco), which contained 10 ng/mL bFGF (Boehringer In- animal in the contralateral ear. One defect was created by gelheim, Ingelheim, Germany) and 20% pooled rabbit serum.
leaving the perichondrium intact, whereas the perichondrium The cells were then plated on T12 culture flasks (Falcon) was excised in the other. The newly created defects were precoated with fibronectin (Sigma, St. Louis, MO). The Cells filled with chondrocytes dissolved in stabilized fibrin gel.
readily adhered to the flask surface and began to spread and In each animal, four defects subjected to four different proliferate after a short lag period. Half the culture medium was changed every other day. After confluency had beenachieved, the cultures were transferred to T75 culture flasks.
• Defect A: No perichondrium, stabilized fibrin, no chondro- • Defect B: Intact perichondrium, stabilized fibrin, no chon- Treatment of Defects With Injectable
Chondrocyte-Fibrin Constructs
• Defect C: No perichondrium, stabilized fibrin, chondro- The first-passage autologous chondrocyte cultures were ready for use after 12 days. Defects for treatment were • Defect D: Intact perichondrium, stabilized fibrin, chondro- created in a new surgical procedure in the contralateral ear.
Two 1-cm2 cartilage segments were again excised, one withand one without the perichondrium. These defects were filled with the stabilized fibrin-chondrocyte solution. After poly- Observation period: Three animals were sacrificed after merization of the solution, the defects were surgically closed.
1, 2, 3, and 6 months after the second operation. Because the Stabilization of commercially available fibrin glue (Immuno, control defects were filled with stabilized fibrin gel already in Vienna, Austria) was achieved by adding 8500 IE/mL apro- the first surgical procedure, a time gap of 12 days existed tinin (Bayer, Leverkusen, Germany) and 15 mg/mL tranex- between control and cell-treated defects. All defects were amic acid (Pharmacia, Stockholm, Sweden) to the solution.
analyzed by histology and immunohistochemistry.
Cells were enzymatically detached from the culture flask,pelleted, and resuspended in the fibrin component of the glue.
Cell density was adjusted to 15 ϫ 106/mL.
Cell Harvesting and Cell Culture
Animals were examined on a regular basis. General After approval by the Austrian Ministry of Science, 12 health status and wound healing were monitored. Thickness female New Zealand White rabbits 1 month old were ob- of operation sites was measured. Three animals were sacri- tained from Charles River and were kept under constant conditions in separate cages. The animals were allowed toacclimate for 3 weeks. Surgery was performed under generalanesthesia using 1.5 mL Ketavet (100 mg/mL) and 0.5 mL Histology
Rompun (2% solution). In the first operation, two 1-cm2 Samples were fixed in a 7.5% buffered formalin solu- segments of auricular cartilage were removed from one ear.
tion and embedded in paraffin. Sections of 4 ␮m were stained One cartilage segment was removed with the perichondrium, with hematoxylin-eosin and Alcian blue for proteoglycan whereas the perichondrium was left intact in the other. The detection. Sections were also stained with orcein for detection defects were filled with stabilized fibrin glue and surgically of elastic fibers. Immunohistochemical staining for collagen II (clone 6B3, Neo Markers, Fremont, CA) was performed on Primary autologous chondrocyte cultures were estab- paraffin-embedded material by the avidin-biotin method. The lished from cartilage segments without the perichondrium.
staining reaction was achieved with 3-amino-9-ethylcarbazol The cartilage segments were washed twice in Hank’s bal- (AEC/Ventana NEXES, Strasbourg, France). Finally, the 2003 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 51, Number 4, October 2003 sections were counterstained with hemalaun (Ventana, Stras-bourg, France).
Evaluation of Tissue Formation and Statistics
Histologic samples were digitized with a high-resolu- tion slide scanner. The amount of cartilaginous and osteoge-nous tissue was calculated from digitized imagines by usingAdobe Photoshop software (Adobe).14,15 For group compar-isons, the Student unpaired t test was performed. Differenceswere considered significant if P Ͻ 0.05.
Wound Healing and Thickness of Treated
FIGURE 1. Histology of defects of type C (no perichondrium,
stabilized fibrin, chondrocytes present) and D (intact perichon- Wound healing appeared to be uneventful in all animals drium, stabilized fibrin, chondrocytes present): Alcian staining.
but one. In this animal, signs of an infection were present in Cut edges are marked by arrows. In defects C and D, the one ear. This animal was successfully treated with antibiotics.
reconstructed cartilage (rc) is clearly visible between the native All type A defects (no perichondrium, stabilized fibrin, cartilage (nc). Digitally enlarged by 50% from scanned Alcian- no chondrocytes) and B defects (intact perichondrium, stabi- lized fibrin, no chondrocytes) were thinner 6 months after theoperation compared with the thickness of the defect areabefore operation.
All type C defects (no perichondrium, stabilized fibrin, chondrocytes present) and type D defects (intact perichon-drium, stabilized fibrin, chondrocytes present) were slightlythicker at 6 months after the operation. (Table 1).
Histology and Immunohistochemistry
After 1 month, histology already revealed cartilaginous tissue in defects treated with stabilized fibrin-chondrocyte (Cand D). The newly formed tissue showed dense cellularityand was directly adjacent to the native cartilage. The cellswere situated in their lacunae and were surrounded by anextracellular matrix (Fig. 1). Moreover, the tissue appeared to FIGURE 2. The reconstructed cartilage (rc) resembled native
be tightly connected to the native tissue (Fig. 2). The amount cartilage (nc) histologically and was tightly connected to it.
of cartilaginous tissue was independent of harvesting date Digitally enlarged by 50% from scanned hematoxylin-eosin– and did not significantly differ between defects of type C and type D (Fig. 3; P Ͼ 0.05). The reconstructed cartilaginoustissue filled between 35% and 90% of type C and D defects(Table 2).
In defects of type A (control defects subjected to only sham treatment), no neocartilaginous tissue formation couldbe detected. In only three animals, there were small areas ofneotissue formation found exclusively at the cut edges of thedefects. The three animals belonged to different analytical groups (1, 2, and 6 months), and the tissue formation did not Thickness Before
Thickness 6 Months
appear to be increasing over time (Figs. 3 and 4, Table 2).
After Surgery
Only minor signs of tissue formation were found in defects oftype B. In this group, areas of tissue formation were found in six animals at the cut edges of the defects. Of these, only two animals showed smaller areas of neocartilage formation in the center of defects, and these animals again belonged to differ- ent analytical groups (1, 2, and 6 months). Smaller areas 2003 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 51, Number 4, October 2003 FIGURE 3. Amount of reconstructed cartilage expressed in number of pixels for defects of type A (no perichondrium, stabilized
fibrin, no chondrocytes), B (intact perichondrium, stabilized fibrin, no chondrocytes), C (no perichondrium, stabilized fibrin,
chondrocytes present), and D (intact perichondrium, stabilized fibrin, chondrocytes present). The amount of reconstructed tissue
in defects C and D appears to be the same at 1, 2, 3, and 6 months. There is no statistically significant difference (P Ͼ 0.05)
between C and D.
Defects, %
FIGURE 4. Histology of defects of type A (no perichondrium,
stabilized fibrin, no chondrocytes) and B (intact perichon- drium, stabilized fibrin, no chondrocytes). Alcian staining. Cut edges are marked by arrows. No neotissue can be seen indefects A and B. In some defects of type B, smaller ossified areas could be detected (arrowhead). Digitally enlarged by 50% from scanned Alcian-stained slides. nc ϭ Native cartilage.
The reconstructed cartilaginous tissue in defects treated of ossification could be detected in some samples from month with stabilized fibrin-chondrocyte constructs stained positive 2 onward in samples of types B, C, and D (Figs. 3 and 4, for collagen II, which was abundant in type C and D defects after 1, 2, 3, and 6 months (Fig. 5). In defects undergoing 2003 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 51, Number 4, October 2003 study also demonstrated that constructs with higher fibrino-gen concentration but low fibrinolytical inhibition were notstable. Constructs with a higher fibrinogen concentration andhigher fibrinolytical inhibition were stable, but matrix pro-duction was reduced compared with constructs with lowfibrinogen concentrations and high fibrinolytical inhibition.
The current in vivo study therefore used stabilized fibrin-chondrocyte constructs. Cartilaginous tissue was foundin all samples derived from defects treated with stabilizedfibrin-chondrocyte constructs. Reconstructed tissue resem-bled native elastic cartilage both histologically and immuno-histochemically. The newly formed cartilage appeared to bedirectly adjacent to the native cartilage.
A recent study demonstrated that cartilage segments joined by a fibrin-chondrocyte solution exhibit substantially FIGURE 5. Immunohistochemistry (Ab vs. collagen II) of the
more tensile strength than those held together by fibrin glue reconstructed cartilage of defects of type C (no perichon- alone.23 This fact is of particular importance in therapeutic drium, stabilized fibrin, chondrocytes present) and D (intact applications, in which the tissue is exposed to continuous perichondrium, stabilized fibrin, chondrocytes present). Colla-gen II is abundant in reconstructed cartilage (rc) and native mechanical forces as in articular, auricular, or nasal septal cartilage (nc). Digitally enlarged by 50% from scanned slides.
Between 35% and 90% of the defected area was filled by cartilaginous tissue. Practical surgical experience shows sham treatment only, areas exhibiting collagen II could be that this is most likely a result of an uneven distribution of the detected only in subjects with limited spontaneous neocarti- solution at the site of the defect. A more reliable injection method is therefore required to ensure that an even recon-struction is achieved.
DISCUSSION
No regeneration or only minor regeneration was ob- Fibrin-chondrocyte constructs have been tested for car- served in defects undergoing sham treatments, even when the tilage reconstruction in vitro and in vivo. In vitro, it has been perichondrium was left intact. The perichondrium also had no shown that fibrin is quickly degraded by chondrocytes.16 In obvious effect on defects treated with stabilized fibrin-chon- vivo, fibrin-chondrocyte constructs polymerized from cryo- drocyte constructs. The amount and morphologic appearance precipitated autologous fibrinogen have been used by some of the reconstructed tissue was identical in samples with and groups for cartilage reconstruction with favorable results,17,18 whereas other groups saw extensive shrinkage19,20 or early A few samples taken during the study exhibited evi- resolution of the fibrin glue and replacement by fibrous dence of ossification. These samples were derived from tissue.21 In an earlier in vitro study by this author,13 it was defects treated either with stabilized fibrin-chondrocyte con- found that fibrin-chondrocyte constructs were dissolved too structs or from defects that underwent only sham treatments early, before cartilaginous tissue formation could occur. It but retained an intact perichondrium. No ossification was could thus be demonstrated that stabilization of chondrocyte- observed in defects in which the perichondrium was removed fibrin constructs by the addition of high concentrations of or in defects that did not have chondrocytes in the fibrin antifibrinolytic substances is a feasible method for three- dimensional formation of cartilaginous tissue in vitro. The The potential of transplanted perichondrium to form use of high concentrations of fibrinolytic inhibitors for stabi- bone has been documented,24,25 and it has also been shown lization slows degradation to an extent that provides enough that isolated chondrocytes can form calcifying cartilage when time for matrix production. Potential systemic and adverse injected into the muscle of animals.26 Bone formation has effects of aprotinin and tranexamic acid include anaphylactic also been seen in immunosuppressed animals after injection reactions, thrombosis, and local thrombophlebitic events. We of allogeneic cultured chondrocytes.27 A recent publication did not observe any of these events in our animals. Moreover, also suggests that hypertrophic chondrocytes can differentiate similar high doses of aprotinin and tranexamic acid have been into osteoblast-like cells contributing to bone formation, but used clinically for the reconstruction of peripheral arteries3 only if they are located at the border of the osteogenic It has also been shown that degradation can be slowed Stabilized fibrin-chondrocyte constructs may therefore by a higher fibrinogen concentration.22 However, the in vitro be useful not only for cartilage reconstruction but also for the 2003 Lippincott Williams & Wilkins Annals of Plastic Surgery • Volume 51, Number 4, October 2003 reconstruction of bone. The evaluation of factors influencing 13. Meinhart J, Fussenegger M, Höbling W, et al. Stabilization of fibrin- the different pathways of tissue formation in fibrin-chondro- chondrocyte construct for cartilage reconstruction. Ann Plast Surg.
1999;42:673– 678.
cyte constructs will be a challenging topic for future studies.
14. Lehr HA, Mankoff DA, Corwin D, et al. Application of Photoshop based image analysis to quantification of hormone receptor expression in ACKNOWLEDGEMENTS
breast cancer. J Histochem Cytochem. 1997;45:1559 –1565.
15. Brunner J, Krummenauer F, Lehr HA. Quantification of video-taped The authors thank Renate Lehner for skillful technical images in microcirculation research using inexpensive imaging software assistance and Ludwig Wallaberger for providing rabbit se- (Adobe Photoshop). Microcirculation. 2000;7:103–107.
rum for cell cultures. The authors acknowledge Steve Rossa 16. Homminga GN, Buma P, Koot HWJ, et al. Chondrocyte behavior in and Joe Ioculano for reading the manuscript.
fibrin glue. Acta Orthop Scand. 1993;64:441– 445.
17. Hendrickson DA, Nixon AJ, Grande DA, et al. Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects.
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2003 Lippincott Williams & Wilkins

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