Zusammenfassung
Die Behandlung von Knorpeldefekten stellt noch immer ein großes Problem in der Orthopädie dar. Eine vielversprechende Alternative zu konventionellen Methoden ist die Reimplantierung von in vitro vorkultivierten Chondrozyten im Rahmen des Tissue Engineering von Knorpel. Die bei diesen Verfahren verwendeten zellbesiedelten Biomaterialien können durch mechanische Stimulation in ihren Regenerateigenschaften verbessert werden. Eine Modulation der Chondrozytenfunktion und dadurch der biologischen und biomechanischen Eigenschaften des Knorpelersatzgewebes lässt sich durch Scher-, Perfusions-, hydrostatische Druck- und direkte Krafteinleitungssysteme erzielen, die beispielhaft und primär in ihren biologischen Auswirkungen vorgestellt werden. Vor dem Hintergrund der trotz günstiger Voraussetzungen und aussichtsreicher Ansätze bisher noch nicht gelungenen In-vitro-Kultivierung von mechanisch und biologisch vollwertigem funktionellem Knorpelersatz werden die Grundlagen, Ergebnisse, Chancen und Probleme der jeweiligen Kultivierungsmodalität beleuchtet und zukünftige Ansätze vorgestellt.
Abstract
The treatment of cartilage defects remains a major problem in orthopaedics. With regard to cartilage tissue engineering, the reimplantation of pre-cultivated chondrocytes in the form of a chondrocyte graft is a promising alternative to conventional methods. Clinical practice requires this MACT procedure (matrix-associated autologous chondrocyte transplantation) to produce a biocompatible replacement tissue with adequate mechanical properties. Mechanical stimulation has the capacity to improve the quality of these cell-seeded biomaterials. By altering chondrocytes' cellular activities, the biological and biomechanical properties of cartilage replacement tissue can be modulated. Different systems are used for this purpose, e.g. shear, perfusion, hydrostatic pressure or compression. The mechanisms, biological effects, chances and problems of the techniques are presented and assessed. Among the stimulating techniques considered are systems that apply indirect and direct shear forces such as spinner flasks, rotating-wall bioreactors, direct tissue shear and perfusion culture systems. The application of hydrostatic pressure or compression may be brought about by either static or dynamic loading systems. Compressive loading is considered in the light of both its short- and long-term effects; additionally two exemplified systems are discussed in detail. However, despite promising approaches and seemingly favourable tissue characteristics, the in vitro culturing of functional cartilage replacement tissue with cartilage-like mechanical and biological characteristics still remains elusive. Furthermore, controlling, monitoring and regulating culturing conditions are general biotechnological requirements of a standardised in vitro cultivation. Among these, different aspects such as aseptic operation, media supplementation, nutrient and gas exchange, temperature and humidity control are considered.
Schlüsselwörter
Tissue Engineering von Gelenkknorpel - zellbesiedelte Matrix - Bioreaktorsystem - Knorpelregeneratgewebe - zyklische Kompression
Key words
tissue engineering of articular cartilage - cell‐seeded scaffold - bioreactor - cartilage replacement tissue - dynamic loading
Literatur
1
Darling E M, Athanasiou K A.
Articular cartilage bioreactors and bioprocesses.
Tissue Eng.
2003;
9
9-26
2
Schulz R M, Bader A.
Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes.
Eur Biophys J.
2007;
36
539-568
3
Eckstein F, Lemberger B, Gratzke C et al.
In vivo cartilage deformation after different types of activity and its dependence on physical training status.
Ann Rheum Dis.
2005;
64
291-295
4
Vunjak-Novakovic G, Freed L E, Biron R J et al.
Effects of mixing on the composition and morphology of tissue-engineered cartilage.
AIChE J.
1996;
42
850-860
5
Vunjak-Novakovic G, Martin I, Obradovic B et al.
Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.
J Orthop Res.
1999;
17
130-138
6
Raimondi M T, Boschetti F, Falcone L et al.
Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment.
Biomech Model Mechanobiol.
2002;
1
69-82
7
Smith R L, Donlon B S, Gupta M K et al.
Effects of fluid-induced shear on articular chondrocyte morphology and metabolism in vitro.
J Orthop Res.
1995;
13
824-831
8
Waldman S D, Spiteri C G, Grynpas M D et al.
Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro.
J Orthop Res.
2003;
21
590-596
9
Waldman S D, Spiteri C G, Grynpas M D et al.
Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.
Tissue Eng.
2004;
10
1323-1331
10
Pazzano D, Mercier K A, Moran J M et al.
Comparison of chondrogensis in static and perfused bioreactor culture.
Biotechnol Prog.
2000;
16
893-896
11
Sittinger M, Bujia J, Minuth W W et al.
Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture.
Biomaterials.
1994;
15
451-456
12
Davisson T, Sah R L, Ratcliffe A.
Perfusion increases cell content and matrix synthesis in chondrocyte three-dimensional cultures.
Tissue Eng.
2002;
8
807-816
13
Mizuno S, Allemann F, Glowacki J.
Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges.
J Biomed Mater Res.
2001;
56
368-375
14
Hall A C, Horwitz E R, Wilkins R J.
The cellular physiology of articular cartilage.
Exp Physiol.
1996;
81
535-545
15
Toyoda T, Seedhom B B, Kirkham J et al.
Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.
Biorheology.
2003;
40
79-85
16
Toyoda T, Seedhom B B, Yao J Q et al.
Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.
Arthritis Rheum.
2003;
48
2865-2872
17
Hall A C, Urban J P, Gehl K A.
The effects of hydrostatic pressure on matrix synthesis in articular cartilage.
J Orthop Res.
1991;
9
1-10
18
Smith R L, Rusk S F, Ellison B E et al.
In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure.
J Orthop Res.
1996;
14
53-60
19
Lammi M J, Inkinen R, Parkkinen J J et al.
Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.
Biochem J.
1994;
304
723-730
20
Takahashi K, Kubo T, Arai Y et al.
Hydrostatic pressure induces expression of interleukin 6 and tumour necrosis factor alpha mRNAs in a chondrocyte-like cell line.
Ann Rheum Dis.
1998;
57
231-236
21
Ikenoue T, Trindade M C, Lee M S et al.
Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro.
J Orthop Res.
2003;
21
110-116
22
Smith R L, Lin J, Trindade M C et al.
Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression.
J Rehabil Res Dev.
2000;
37
153-161
23
Parkkinen J J, Ikonen J, Lammi M J et al.
Effects of cyclic hydrostatic pressure on proteoglycan synthesis in cultured chondrocytes and articular cartilage explants.
Arch Biochem Biophys.
1993;
300
458-465
24
Heath C A, Magari S R.
Mini-review: mechanical factors affecting cartilage regeneration in vitro.
Biotechnol Bioeng.
1996;
50
430-437
25
Carver S E, Heath C A.
Increasing extracellular matrix production in regenerating cartilage with intermittent physiological pressure.
Biotechnol Bioeng.
1999;
62
166-174
26
Mizuno S, Tateishi T, Ushida T et al.
Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture.
J Cell Physiol.
2002;
193
319-327
27
Tanck E, van Driel W D, Hagen J W et al.
Why does intermittent hydrostatic pressure enhance the mineralization process in fetal cartilage?.
J Biomech.
1999;
32
153-161
28
Elder B D, Athanasiou K A.
Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration.
Tissue Eng Part B Rev.
2009;
15
43-53
29
Horowitz S B, Lau Y T.
A function that relates protein synthetic rates to potassium activity in vivo.
J Cell Physiol.
1988;
135
425-434
30
Elder B D, Athanasiou K A.
Synergistic and additive effects of hydrostatic pressure and growth factors on tissue formation.
PLoS One.
2008;
3
e2341
31 Gooch K J, Tennant C J. Chondrocytes.. Mechanical Forces: their Effects on Cells and Tissues.. Georgetown, TX, USA: Landes Bioscience; 1997: 79-100
32
Guilak F, Ratcliffe A, Mow V C.
Chondrocyte deformation and local tissue strain in articular cartilage: a confocal microscopy study.
J Orthop Res.
1995;
13
410-421
33
Buschmann M D, Gluzband Y A, Grodzinsky A J et al.
Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture.
J Cell Sci.
1995;
108
1497-1508
34
Guilak F.
Compression-induced changes in the shape and volume of the chondrocyte nucleus.
J Biomech.
1995;
28
1529-1541
35
Lee C R, Grodzinsky A J, Spector M.
Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression.
J Biomed Mater Res A.
2003;
64
560-569
36
Lee D A, Bader D L.
Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose.
J Orthop Res.
1997;
15
181-188
37
Mauck R L, Soltz M A, Wang C C et al.
Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.
J Biomech Eng.
2000;
122
252-260
38 Mansour J. Biomechanics of Cartilage.. Kinesiology: the Mechanics and Pathomechanics of Human Movement.. Baltimore: Lippincott Williams and Wilkins; 2004: 66-79
39
Lee D A, Noguchi T, Frean S P et al.
The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs.
Biorheology.
2000;
37
149-161
40
Kisiday J D, Jin M, DiMicco M A et al.
Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.
J Biomech.
2004;
37
595-604
41
Davisson T, Kunig S, Chen A et al.
Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage.
J Orthop Res.
2002;
20
842-848
42
Wang P Y, Chow H H, Tsai W B et al.
Modulation of gene expression of rabbit chondrocytes by dynamic compression in polyurethane scaffolds with collagen gel encapsulation.
J Biomater Appl.
2009;
23
347-366
43
Hunter C J, Imler S M, Malaviya P et al.
Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels.
Biomaterials.
2002;
23
1249-1259
44
Mauck R L, Byers B A, Yuan X et al.
Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.
Biomech Model Mechanobiol.
2007;
6
113-125
45
Demarteau O, Wendt D, Braccini A et al.
Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.
Biochem Biophys Res Commun.
2003;
310
580-588
46 Lee C, Grad S, Wimmer M A et al. The Influence of Mechanical Stimuli on articular Cartilage Tissue Engineering.. Topics in Tissue Engineering.. Oulu: Biomaterials and Tissue Engineering Group; 2006: 1-32
47
Huang C Y, Hagar K L, Frost L E et al.
Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells.
Stem Cells.
2004;
22
313-323
48
Elder S H, Kimura J H, Soslowsky L J et al.
Effect of compressive loading on chondrocyte differentiation in agarose cultures of chick limb-bud cells.
J Orthop Res.
2000;
18
78-86
49
Schulz R M, Wustneck N, van Donkelaar C C et al.
Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs.
Biotechnol Bioeng.
2008;
101
714-728
50
Burton-Wurster N, Vernier-Singer M, Farquhar T et al.
Effect of compressive loading and unloading on the synthesis of total protein, proteoglycan, and fibronectin by canine cartilage explants.
J Orthop Res.
1993;
11
717-729
51
Frank E H, Grodzinsky A J, Koob T J et al.
Streaming potentials: a sensitive index of enzymatic degradation in articular cartilage.
J Orthop Res.
1987;
5
497-508
52
Takahashi I, Nuckolls G H, Takahashi K et al.
Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.
J Cell Sci.
1998;
111
2067-2076
53
Sah R L, Kim Y J, Doong J Y et al.
Biosynthetic response of cartilage explants to dynamic compression.
J Orthop Res.
1989;
7
619-636
54
Steinmeyer J.
A computer-controlled mechanical culture system for biological testing of articular cartilage explants.
J Biomech.
1997;
30
841-845
55
Steinmeyer J, Torzilli P A, Burton-Wurster N et al.
A new pressure chamber to study the biosynthetic response of articular cartilage to mechanical loading.
Res Exp Med (Berl).
1993;
193
137-142
56
Sauerland K, Raiss R X, Steinmeyer J.
Proteoglycan metabolism and viability of articular cartilage explants as modulated by the frequency of intermittent loading.
Osteoarthritis Cartilage.
2003;
11
343-350
57
Steinmeyer J, Knue S.
The proteoglycan metabolism of mature bovine articular cartilage explants superimposed to continuously applied cyclic mechanical loading.
Biochem Biophys Res Commun.
1997;
240
216-221
58
Angele P, Yoo J U, Smith C et al.
Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro.
J Orthop Res.
2003;
21
451-457
Sven Nebelung
Klinik für Orthopädie und Unfallchirurgie Universitätsklinikum Aachen
Pauwelsstraße 30
52057 Aachen
Telefon: 02 41/8 08 55 85
Fax: 02 41/8 08 24 53
eMail: sven.nebelung@rwth-aachen.de