Artificial chondrons incartilage tissue engineering

A preliminary study

K.H. ter Stege

Report no: BMTE07.04

TU/e Internship Report

16th February 2007

Supervisors:K. Ito M.D. Sc.D. (AO)dr. C. C. van Donkelaar (TU/e)

Eindhoven University of Technology AO Research InstituteDepartment of Biomedical Engineering Mechano-biology program

Division Materials Technology Davos - Switzerland

Contents

Introduction v

Background 11.1 Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.2 Proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.3 Collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2 Chondron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.3 Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.4 PUR-scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.5 Fibrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Materials & Methods 72.1 Isolating chondrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.2 PUR-scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2.1 Properties of the PUR-scaffolds . . . . . . . . . . . . . . . . . . . . . . . 92.2.2 Seeding the PUR-scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3 Seeding the alginate beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.4 Dissolving the alginate beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.5 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.5.1 Preparation of the samples . . . . . . . . . . . . . . . . . . . . . . . . . . 122.5.2 DNA-assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.5.3 GAG-assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.5.4 OHP-assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.6 Viability assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.7 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.7.1 Preparation of the samples . . . . . . . . . . . . . . . . . . . . . . . . . . 142.7.2 Alcian blue stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.7.3 Hematoxylin/ Safranin O/ Fast Green stain . . . . . . . . . . . . . . . . . . 142.7.4 Toluidine blue stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.7.5 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.8 Statistical evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

iii

iv

Results 173.1 General observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.2 Assay results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173.3 Histology results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Discussion & Conclusion 29

Recommendations 31

References 37

Introduction

Articular cartilage is an important mechanical entity covering the joint surfaces. Damaged carti-lage often results in progressive osteoarthritis, because cartilage has a very low self-repairing orself-regenerating capacity [40]. In order to treat osteoarthritis, joint resurfacing, autograft trans-plants, prosthesis and complete joint replacement have been evaluated [27][47]. Unfortunately,these methods are all limited in their repair capabilities and usability.

When tissue engineering came up in the late nineties, tissue engineering of cartilage was fromscratch one of the main topics. It is advantageous to work with cartilage, since it is a relativesimple tissue consisting only of chondrocytes and their extracellular matrix (ECM). Furthermorecartilage is avascular, so chondrocytes stay viable in a non-vascularized environment and it hasa low immunogenicity.

Various approaches of cartilage tissue engineering have been tested throughout the years. Cellshave been cultured in monolayers, micromasses or scaffolds [13][22][46]. Scaffolds show themost promising results, so many different types of scaffolds have been developed and tested:

• hydrogels, such as alginate, agarose and fibrin [10][34][43]

• natural polymers, such as collagen and chitosan [21][31]

• biodegradable materials such as polyurethane, polylactides, hydroxyapatite and polyglycol-ide [6][16][45][50]

To enhance cartilage matrix synthesis, continuous or periodic loading [8][29][49], different typesof bioreactors [47] and different oxygen-tensions [41][51] have been evaluated. Until today noneof these methods has created a cartilage-like structure with the exact same composition, appear-ance and mechanical properties as normal cartilage.

Chondrocytes cultured in a scaffold do not show a homogenous matrix production throughoutthe scaffold. Matrix production is mostly observed around the edges of the scaffolds [19][28][42].The cells at the boundaries of the construct use all available nutrients, so less nutrients enterthe scaffold. Nutrient availability is required for matrix synthesis. Hence, nutrient deficiency in thecenter causes an even less homogenous matrix distribution and eventually cell-dead in the center.

v

vi

The maximal nutrition diffusion-depth determines the maximal dimensions of a construct duringcartilage tissue engineering [7][12]. This maximal diffusion-depth is basically an equilibrium be-tween nutrient diffusion according to Fick’s law and the nutrition consumption of the cells. Thenutrient concentration in the center of a construct rises when cells consume smaller amounts ofnutrients. A difference in nutrient consumption between a single cell and a chondron (cell sur-rounded by its pericellular matrix (PCM)) is expected, because cells need to generate a PCM anda ECM, while chondrons only need to generate an ECM [54].

Natural chondrons appear throughout the thickness of cartilage with differences in size, shapeand cell-number [39][53]. They can also be cultured artificially in an alginate bead where thealginate can be dissolved to extract the chondrons [20][33]. These artificial chondrons are moreuniform than natural chondrons, since they have all been grown in a nutrient-rich environment.

It is hypothesized that when chondrons are cultured in a poly-urethane-resorbable (PUR)-scaffoldthe matrix produced will be more homogenous and more viable cells will remain in the center ofthe construct. The following question is addressed in this report to test this hypothesis:

Does a scaffold seeded with artificial chondrons produce a more homogenous matrixwith more viable cells than a scaffold seeded with single chondrocytes?

Primary chondrocytes harvested from a bovine fetlock joint are cultured in two ways. First, they areseeded in an alginate hydrogel for 10 days to create chondrons. These chondrons are transferredto a PUR-scaffold. Second, primary chondrocytes are seeded immediately in a similar PUR-scaffold. After 21 days of culture matrix-production and viability is determined. The PUR-scaffoldhas been chosen since this in-house developed scaffold has given very promising results so farin cartilage tissue-engineering [14][16].

Background

1.1 Cartilage

Articular cartilage is a highly specialized, avascular, aneural, connective tissue that enables easyjoint movement [4]. Cartilage is built of several components of which the main component is water,which can reach levels between 65 and 80% in healthy cartilage. The dry weight contains mainlycollagens (60%) and proteoglycans (30%) and the last 10% consists of glycoproteins, lipids andchondrocytes [4][35].

Articular cartilage can be described as glassy smooth, glistening structure that is blueish-whitein appearance. It contains a superficial, an intermediate, a deep and the calcified layer. Notonly the shape, size and clustering of the cells differs throughout these layers, the composition ofthe ECM is also not the same. The water content of cartilage is highest at the superficial layer(around 80%), but near the calcified layer it has decreased to 65%. The collagen content alsodecreases throughout the layers, from 85%dw in the superficial layer to 65%dw in the deep layer.The proteoglycan content is highest in the middle layer and decreases towards the top and bot-tom of the cartilage. The amount of proteoglycans in the deep zone of cartilage is higher than theproteoglycan content in the superficial zone (table 1.1).

Table 1.1: Summary of the ECM distribution within the full thickness of cartilage

Thickness Water Collagen Proteoglycanof layer content content content

Superficial zone 10-20% ≈ 80% ≈ 85% (dry weight) lowestMiddle zone 40-60% highestDeep zone 20-25% ≈ 65% ≈ 65% (dry weight) middle

Calcified zone 5-10%

1

2

Figure 1.1:Matrix definitions around chondrocytes; left picture from Poole [36], ’C’ is the chondrocyte, ’Pg’ showsthe pericellular matrix and ’Pc’ the pericellular capsule. Right picture from University of Oklahoma [56]

1.1.1 ChondrocytesChondrocytes are the only cells that appear in adult cartilage. Chondrocytes are round or polygo-nic in shape, but can be flattened near the edge of the tissue [2]. Chondrocytes synthesize andmaintain the matrix of collagens and proteoglycans in which they are situated [23]. They commu-nicate through paracrine signaling and produce their matrix at a low oxygen tension (ranging from10% at the edge to <1% in the deep zone of articular cartilage), since cartilage is avascular.The matrix closest to the chondrocyte is the pericellular matrix (PG in figure 1.1). The perichon-drium or pericellular capsule (PC in figure 1.1) separates the pericellular matrix from the territorialmatrix. Chondrocytes in the middle and deep layers of cartilage are well integrated with their terri-torial matrix (right part of figure 1.1). Matrix that is not integrated with any chondrocyte is definedas the interterritorial matrix [36].

1.1.2 ProteoglycansAll proteoglycans are macromolecules composed of glycosaminoglycan (GAG) carbohydrate chains,which are covalently bounded to a protein core. There are 2 types of proteoglycans (PG) in artic-ular cartilage: Large aggregating PG’s like aggrecan (80-90%) and small PG’s like biglycan anddecorin [4]. Biglycan has a core of 40 kDa and a single chondroitin sulfate chain. It is found in theinterterritorial matrix, while decorin is found in the pericellular matrix. Decorin has a similar corebut a double chondroitin sulfate chain [4]. Decorin is important for collagen fibril formation andhas the ability to immobilize certain growth factors. This indicates that decorin regulates matrixproduction [35]. Aggrecan consists of a core protein with chondroitin sulfate and keratan sulfateside-chains. Numerous aggrecan monomers aggregate and attach non-covalently to hyaluronan,stabilized by link protein [35].The side-chains of PG’s contain a lot of negative charges, so the osmotic pressure is high and wa-ter integrates between the aggregates. PG’s are able to carry 50 times their own weight (70% oftotal water content). Due to this water the aggregates swell and produce the pressure-resistanceknown for cartilage [23]. The interactions between proteoglycans, collagens and water give thematrix a low hydraulic permeability. This results in a minimal loss of fluid when the cartilage isloaded [32].

342 CURRENT ORTHOPAEDICS-BASIC SCIENCE

Proteoglycan & Water

Hyaluronic Acid

Fig. 6-Proteoglycan aggregate and interaction with water represented diagrammatically.

described as forming a dense network with fibres running obliquely between the articular surface and the subchondral bone. MacConaill also noted a thin bright line at the articular surface representing a surface layer which appeared devoid of collagen fibres and which he named the ’ lamina splendens ‘. This was regarded as a hardened or chondrified continuation of the synovial membrane onto the surface of the

joint,~0,"l,'"

Using scanning electron-microscopy and freeze- fracture techniques a three-dimensional organisation of collagen has been proposed with organisation in a layered or leaf-like manner” [Fig. 5A). In the superficial zone the leaves were arranged in a horizontal manner parallel to the articular surface, curving in the intermediate zone to become more vertically orientated. In the superficial and inter- mediate layers the collagen leaves were noted to consist of a fine network or mesh of Type II collagen fibrils (Fig. 5B). In the deeper zones the fibres were coarser and organised in a more vertical and columnar manner. The organisation of collagen in a 3- dimensional manner such as this would explain the anisotropic properties of cartilage where the mech- anical properties of cartilage have been noted to differ depending on the direction of loading and the zone of cartilage being examined. It could also provide an explanation for the well-known split-line phenomenon where articular cartilage pricked by a round-bodied pin instead of producing round holes, forms characteristic and constant split-line patterns in the c-.rtilage.lO.li.23

3. ProteoglJv-an

The proteoglycans in articular cartilage are large protein-polysaccharide molecules. The proteoglycan monomer is composed of keratan sulphate and chondroitin sulphate chains bound covalently to a protein core molecule. The monomers may be visual- ised as having a bottle-brush-like structural arrange- ment with the keratan sulphate and chondroitin sulphate (glycosaminoglycans or GAGS for short) attached to, and radiating perpendicularly from, the

Proteo$lycan Aggregate

Collagen

Fig. 7-The meshwork of Type II collagen fibrils constraining and interacting with the proteoglycan aggregates. The proteoglycan bound with water creates a swelling tendency which pre-stresses the collagen and gives cartilage its characteristic elasticity.

protein core (Fig. 6). Smaller non-aggregating proteo- glycan molecules containing dermatan sulphate are also seen.” In articular cartilage most of the proteo- glycan monomers are associated with hyaluronate which is a derivitive of hyaluronic acid, a large polymer of repeating identical disaccharide units.“’ The proteoglycan and hyaluronate combine to form large proteoglycan-hyaluronate aggregates. A link protein binds simultaneously to both the hyaluronic acid and the proteoglycan monomer to stabilise the aggregate (Fig. 6). The aggregate may have a molecular weight as high as 200 million and the filamentous backbone of hyaluronic acid is of the order of 200 nm in length. Like the collagen, all the components of the proteoglycan aggregate are synthe- sised by chondroblasts and chondrocytes and largely undergo self-assembly in an extra-cellular situation.

4. Structural interaction between collagen, proteoglycan and Ii’ater

In solution, the electro-negative charges on the sulphated sugars of the proteoglycan cause the monomer side chains to repel one another and to attract water (Fig. 6). The meshwork of collagen fibrils forms a honeycomb or series of compartments in which the proteoglycan complexes are compressed to about one-fifth of their maximal volume in free solution (Fig. 7).“.”

The collagen fibrils and the proteoglycan have a close physical relationship and the interaction between the proteoglycan and the collagen network has a direct role in the organisation of the matrix. This relationship also contributes directly to the mech- anical properties of articular cartilage.3 Electrostatic interactions and physical entanglements may exist

Figure 1.2:Integration of water inside articular cartilage [23]

1.1.3 Collagens

Articular cartilage mainly consists of collagen type II (90-95%), which is a fibril-forming collagen.Other collagens are the beaded filament-forming collagens (type VI) and fibril-associated colla-gens with interrupted triple helices (types IX, XII, XIX) [35]. Collagens consist of three polypeptideα chains which form a left-handed coiled matrix. Together these 3 α-chains form a right-handedtriple helix [23], [35].Collagen II in cartilage enables resistance against osmotic swelling pressure created by the highlycharged proteoglycans, since collagen fibers are very strong in tension [4]. Collagens are pre-stressed by water bonded to the proteoglycans resulting in the characteristic elasticity known forcartilage [23].

1.2 Chondron

The concept of a ”chondron” was first introduced by Benninghoff in 1925 [3]. He described thestructural unit of a cel/ cell group in cartilage together with its immediately surrounding matrix.This definition is specified a little over time. The chondron is now defined as a capsule separatingthe chondrocyte(s) and its pericellular matrix from the territorial matrix [37].Chondrons can contain one or more cells (multi-cellular appears only in the middle and deep layerof cartilage) [38]. Superficial chondrons are discoidal, in the middle zone they are more roundedand in the deep zone they have a more cocoon-shaped form (figure 1.3) [53].

4

Figure 1.3:Left side of the picture shows the shape of the chondrons from the different layers of cartilage [53] andthe right side shows the shape of a deep-zonal chondron [36]

The cocoon-shaped deep-zonal chondrons show a dense compaction of pericellular collagens atthe articular pole (AP in figure 1.3) and a loosely woven tail tapering to the basal pole (BP in figure1.3). When a deep-zonal chondron consists of more than one cell this cocoon form can still befound and in those cases the tails form interconnecting segments between adjacent chondronsand ensure linear continuity between adjacent chondrocytes [36].

The chondron has a mechanical and a metabolic function in cartilage. The chondron can absorbmechanical load by deforming laterally or shear [3][5][36][44]. When the load is removed, thechondron will recover completely, so it is proposed that the chondron is a micro-mechanical unitin which the exaggerated swelling pressure is restricted by the capsular collagens. Chondronshave metabolic role in the spatial and temporal differentiation of newly synthesized aggrecan andthey play a role in the retention, maturation, differentiation and turnover of aggrecan [36].

1.3 Alginate

Alginate is a polysaccharide that can be found in all brown marine algae (Phaeophyceae) and insome microorganisms [52]. It is a linear copolymer composed of β− (1,4)-linked D-mannuronicacid (M-component) and α− (1,4)-linked L-guluronic acid (G-component).Alginate is not a random copolymer but consists, according to the source algae, of blocks ofsimilar and strictly alternating residues [58]. The alginate used has long M-blocks (see figure 1.5),with smaller G-blocks in between.Gels formed from an alginate with high amounts of G-blocks show higher mechanical strength,lower shrinkage, higher porosity and more stability towards monovalent cations [52]. High amountsof M-blocks result in more elastic gels and formed in presence of Ca2+ high amounts of M resultin stronger gels [58].

5

O

OH

OH

OHOH

COO-

O

OH

OH

OHOH

COO-

Figure 1.4:β− (1,4)-linked D-mannuronic acid or M and α− (1,4)-linked L-guluronic acid or G [48]

O

O

O

O

HOHO

HOHO

HO

COO-

COO-

COO-

COO-

O

O

O

O

HO HOHO

O

O

O

O

O

O

O

O

COO-

COO-

COO-

COO-

OH

OH

OH

OH

OH

OH

OH

OH

Figure 1.5:Upper drawing shows a polymer of G-blocks and lower shows a polymer of M-blocks [57]

When calcium ions are added to a sodium alginate solution, two aligned G-block regions showdiamond shaped holes. The calcium ions are trapped inside these holes like eggs in an egg box(figure 1.6) and a gel is formed [18][57]. This calcium can be removed without damaging the cells,by adding a buffer containing Sodium-citrate and EDTA, since these components show a higheraffinity for Ca2+ than the alginate does.

1.4 PUR-scaffold

The use of a scaffold is essential for support and augment of the initial cell growth and differenti-ation during tissue engineering. Ideally a scaffold should fulfill the following conditions:

• Composed of a biocompatible material avoids rejection by host

• Degradation (in non-toxic molecules) provides space for newly generated tissue

• Provide sufficient initial strength to support the injured tissue

Polyurethanes are useful in tissue engineering. Points in favor of polyurethanes are durability,elasticity, elastomer-like character, fatigue resistance, compliance, degradation via hydrolysis andtolerance in the body. Polyurethanes also have the possibility to adjust hydrophobic/ hydrophilicbalance and to build in biologically active molecules (functional groups) [1].

6

G-block

regions

Gel

Solution

+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+ Ca

2+

Fibrinogen

Fibrin monomer

Fibrin dimer

Fibrin polymer

Thrombin Fibrinopeptide A

Fibrinopeptide B

Figure 1.6:Schematic drawing of the egg-box model [57] Figure 1.7: Schematic polymerization process of fibrino-gen to fibrin [55]

Depending on their mechanical properties, chemical composition and surface characteristicsbiodegradable polyurethanes can potentially be used for cardiovascular implants, drug deliverydevices, nonadhesive barriers in trauma surgery, cancellous bone graft substitutes, tissue engi-neering scaffolds, or adhesives [14].

1.5 FibrinFibrin is a natural polymer that is made up from fibrinogen and prothrombin in the presence ofcalcium. The calcium converts the prothrombin to thrombin, which activates the fibrinogen. Ac-tive fibrinogen is called a fibrin monomer. These monomers polymerize at body temperature andform the fibrin polymer (see figure 1.6) [55]. Fibrin has a bioactive role within the human bodythrough specific receptor-mediated interactions with cells. It is available in the ECM and involvedin hemostasis and wound healing. Fibrin is formed at sites of tissue injury and provides a tempo-rary matrix to support the initial response of endothelial epithelial and mesenchymal cells neededfor tissue repair [9].

In tissue engineering fibrin is used as a scaffold for cartilage tissue engineering, since chondro-cytes maintain their phenotype in this three-dimensional environment [9]. Fibrin has been usedto get a better understanding of cell behavior, since the resulting fibrin matrices mimic the in vivomicro-environment in wound healing and other pathologies.Fibrin is also used in combination with the PUR-scaffold described before. It is used as a glueto assure a more homogenous cell-distribution and to improve cell and ECM-molecule retention[28].

Materials & Methods

A set of experiments was scheduled to examine the hypothesis defined in the introduction:

Does a scaffold seeded with artificial chondrons produce a more homogenous matrix with moreviable cells than a scaffold seeded with single chondrocytes?

At day 0 chondrocytes were harvested from a bovine fetlock joint. These chondrocytes were splitup in two groups and half of them were seeded in PUR-scaffolds (5 million cells/ scaffold) and theother half was seeded in alginate beads (40.000 cells/ bead). After 10 days of culture the alginatebeads were dissolved and the chondrons were seeded in fresh PUR-scaffolds. Chondrons werenot harvested immediately from the cartilage because, the chondrons will be grown from mes-enchymal stem cells in the future. After 21 days (from harvesting) both experiments were endedand the scaffolds were examined for differences in DNA, GAG and collagen content. This wasdone with help of biochemical assays and histological evaluation. The time-line shown in Figure2.1 shows the set-up:

Day 0 Day 1 Day 10 Day 21

Harvestchondrocytes

Harvestchondrocytes

Seed cellsin alginate

Seed cells In PUR

Dissolve chondronsfrom alginate and

seed these in PURFix and process

samples

Fix and processsamples

Figure 2.1:Time-line of the experimental set-up

On the next page an overview of all techniques used at each time-point is given after which allthese techniques are presented one by one in more detail.

7

8

Overview of the experimental set-up

Chondrocytes in PUR

Day 0:

• Amount of living cells versus dead cellsTrypan blue stain for counting and theamount of living and dead cells

Day 2 (n = 3):

• Distribution of cellsDNA-, GAG- and hydroxyproline-assaysto see if all matrix was dissolvedSafranin O, Fast Green, Hematoxylin stainto visualize distribution

Live/ dead

stain

DNA/ GAG & OHP/assay

Histology

Day 21 (n = 8):

• Distribution and amount of living ver-sus dead cellsCalcein AM/ Ethidium homodimer stain ona quarter of the unfixed scaffold

• Matrix production (how much and whattype)DNA-, GAG- and hydroxyproline-assaysImmunohistochemistry for GAG, link pro-tein and collagen II to visualize matrix pro-ductionSafranin O, Fast Green, Hematoxylin andtoluidine blue stain to visualize distributionand overall matrix production

Chondrocytes in alginate →Chondrons in PUR

Day 2: xDay 10:

• Chondron distributionAlcian blue stain to check the distributionof the cellsDNA-assay to quantify the amount ofbeads that need to be dissolved

Day 12, 2 days after seeding in PUR (n = 3):

• Distribution and amount of living ver-sus dead cellsCalcein AM/ Ethidium homodimer stain ona quarter of the unfixed scaffold

• Distribution of cellsDNA-, GAG- and hydroxyproline-assaysto determine starting pointSafranin O, Fast Green, Hematoxylin stainto visualize distribution

Day 21 (n = 8):

• Distribution and amount of living ver-sus dead cellsCalcein AM/ Ethidium homodimer stain ona quarter of the unfixed scaffold

• Matrix production (how much and whattype)DNA-, GAG- and hydroxyproline-assaysImmunohistochemistry for GAG, link pro-tein and collagen II to visualize matrix pro-ductionSafranin O, Fast Green, Hematoxylin andtoluidine blue stain to visualize distributionand overall matrix production

9

2.1 Isolating chondrocytes

Chondrocytes were harvested from a fetlock joint. The complete lower leg of a calf (approximately3-4 months old) was collected at the local slaughterhouse (Stiffler, Davos). In a semi-sterileenvironment the skin and tissue were removed from the joint, but it was made sure that the jointcapsule was kept closed. After transfer into a Laminar Air Flow (LAF)-cabinet the joint was openedand the full-thickness cartilage was removed with a fresh sterile scalpel-blade. The cartilage wascut into small pieces and stored in Tyrrode’s Balanced Salt Solution, TBSS. Cartilage pieces weretransferred into a spinner flask (1 flask per joint) and washed three times for 15 minutes with 60mlTBSS containing 10x Penicillin/ Streptomycin (Gibco). Finally the cartilage got pre-digested with a0.1% Pronase-solution (Roche) for 2 hours. After washing 3 times 5 minutes with 60ml plain TBSSthe complete matrix was removed by digestion overnight with collagenase II (Worthington). Singlechondrocytes were filtered out of the solution with a 40µmcell strainer. The cells were spinneddown at 565G for 7 minutes at 4◦C. Medium (high-glucose DMEM supplemented with 10% FetalCalf Serum (FCS)) was added and the cells were washed (centrifuged and re-suspended) twicemore. Cells were counted with a haemocytometer and trypan blue, also determining viability.

2.2 PUR-scaffolds

2.2.1 Properties of the PUR-scaffolds

The scaffolds used in this report were developed in-house (AO Research Institute, Davos). Thescaffolds were synthesized from hexamethylene diisocyanate, poly(ε-caprolactone)diol with amolecular weight of 530 Da and isosorbide diol (1,4:6,6-dianhydro-D-sorbitol) was used as chainextender [15]. The complete process has been described in literature by Gorna et al. in 2000 [14]and is shown in a reaction scheme in Figure 2.2.

2 O=C=N-R-N=C=O + HO-(R’)n-OH → O=C=N-R-NH-CO-O-(R’)N-O-OC-HN-R-N=C=ODiisocyanate Polyol Prepolymer (macrodiisocyanate)

O=C=N-R-NH-CO-O-(R’)n-O-OC-HN-R-N=C=O + HO-R’’-OH (-NH2; -SH) →

Preploymer Chain extender

-[-O-R’’-O-OC-HN-R-NH-CO-O-(R’)n-O-OC-HN-R-NH-CO-O-R’’-O-]x-

Polyurethane

Figure 2.2:Molecular mechanism of polymerization of polyurethanes [15]

10

Figure 2.3:Scanning electron microscopy pictures of the used PUR-sponges. Pictures taken by Markus Glarner(AO Research Institute)

The scaffolds had a low content of the hydrophilic component, allowing adhesion and prolifer-ation of various cell types [15]. This scaffold has been used in 2003 for the first time to seedchondrocytes [16]. The results from Grad et al. were very promising but also showed some lim-itations. These limitations were mostly solved when the chondrocytes were seeded with help offibrin ”glue” as proposed by Lee et al.[28]. It has been shown that this method assures a morehomogeneous distribution of the cells over the scaffold, a higher cell-viability and new matrix is nolonger discarded to the culture medium but adheres inside the scaffold.

The scaffolds were cylindrical (8mm in diameter and a 4mm thickness) with interconnected poresthat showed an average pore-size of 400-600µm (see figure 2.3). The scaffolds were sterilizedusing a cold-cycle (37◦C) ethylene oxide process for 4 hours and were subsequently evacuatedin high vacuum for 5 days to remove the ethylene oxide residues. The molecular weight of thescaffolds was determined to be 70.540Da, which is done with help of the Mark-Houwink equation:

η = K·Mαw

with K = 6.8·10−5 and α = 0.86

The intrinsic viscosity was measured in DMF (dimethyl formamide) at 30.0◦C with a viscometer(Ubbelohde). Measurements have been performed by Markus Glarner (AO Research Institute).

2.2.2 Seeding the PUR-scaffolds

The scaffolds were hydrophobic, and to reduce this hydrophobicity they were put in vacuum 30minutes before use. The scaffolds were seeded with cells mixed with fibrin and this mixture wasput in the lid of an eppendorf-tube. These lids had exactly the same size as the scaffold andallowed the fibrin to be sucked into the scaffold (due to capillary forces) before the polymerizationprocess of te fibrin was completed.

11

The cells were centrifuged into a pellet and mixed with the 1:3 diluted fibrinogen (Baxter bio-surgery). A final concentration of 5 million cells/75µl was obtained. This mixture was placed intothe lid of the eppendorf-tube. Next also 75µl of the 1:500 diluted thrombin (Baxter biosurgery)was added and mixed with the cell-fibrin mixture by gently pipetting up and down. The scaffoldwas put in the polymerizing fibrinogen-cell-thrombin mixture and left in to absorb the mixture. Thewhole combination was placed in the incubator for 60 minutes in order to let the polymerizationproces finish. The scaffolds were taken out of the lid and put in a 12-well plate, one scaffold perwell, with 3ml DMEM (high-glucose) supplemented with 10% FCS per scaffold, 60µg/ml ascor-bic acid (Sigma A8960), 1% aprotinin (Fluka 10820) and 1% non-essential amino-acids (Gibco11140-035). The medium was changed three times a week and the well-plate was changed everyweek to prevent a monolayer formation.

2.3 Seeding the alginate beads

The single chondrocytes were pelleted down and mixed with a 1.2% alginate in 0.9% NaCl solution(Fluka 71240 & Fluka 71380). A final concentration of 4 million cells/ml alginate was obtained. Theplunger of a 10ml syringe was removed and the alginate-/ cell-mixture was pipetted in. A 22G 11

4”(Microlance) needle was attached to the front of the syringe. The beads were formed by droppingthe alginate/cell- mixture in 150ml 102mM CaCl2 (Fluka 21075) solution with a frequency of onedrop per second. The newly formed beads were left in CaCl2 solution for 10 minutes to gelateproperly and were washed three times in 0.9% NaCl and twice in plain DMEM. The beads werecultured in 6-well plates with 30 beads per well and 6ml of DMEM supplemented with 10% FCSand 60µg/ml ascorbic acid (Sigma A8960). Medium was changed every 2-3 days and well-plateswere changed weekly, to prevent monolayer formation.

2.4 Dissolving the alginate beads

The beads were put directly in 150µl/bead (a little over 10 volumes) of a dissolution/digestionbuffer, consisting of 150mM NaCl (Fluka 71380), 55mM Na3Citrate·2H2O (Sigma S1804), 5mMNa2EDTA·2H2O (Sigma E5513), 5mM Cystein-HCl (Fluka 30120) and 125µg/ml Papain (SigmaP3125), dissolved in deionized water. The beads were incubated at 56◦C for 1 hour and gentlyagitated for one minute every ten minutes. The chondrons were centrifuged into a pellet for 15minutes at 565G at 4◦C. The dissolving buffer was removed and the pellet was resuspended withthe same amount of dissolution/digestion buffer and agitated for five full minutes, to make surethat all the alginate was dissolved. The chondrons were centrifuged for 5 minutes at 565G at 4◦C.After removal of the buffer, the pellet of chondrons was ready for use in the further experiments.

2.5 Assays

For the quantitative evaluation of the samples, several assays have been performed. The alginatebeads were examined for their DNA-content, as a method to determine the amount of living cellsin a construct. The scaffolds were evaluated for their DNA-content, their GAG-content and theirOHP-content (=collagen-content).

12

2.5.1 Preparation of the samples

Alginate beads

To determine how many beads needed to be dissolved for further experiments the amount ofcells present in 1 bead after 10 days of culture had to be known. Alginate interferes with theHOECHST-dye used during the DNA-assay, so the alginate beads needed to be dissolved beforethe DNA-assay was performed. Trials had shown that at least two beads needed to be dissolved inorder to generate reliable data. Five times two beads were dissolved as described in the previoussection.

PUR-scaffolds

To half of each scaffold 1ml proteinase K (0.5 mg/ml, Roche) in phosphate buffer was added.The samples were digested overnight at 56◦C. The residue of the digested sample was stored at-20◦C and thawed before use in DNA-, GAG- and OHP-assays.

2.5.2 DNA-assay

The amount of DNA was evaluated using bisBenzimide Hoechst-33528 fluorescent dye (Fluka14530), prepared from a stock solution to a concentration of 1µg/ml in DPBS (25mM KH2PO4,25mM Na2HPO4, 2.0M NaCl). The amount of DNA was measured against a standard curveconsisting of a known amount of calf thymus DNA (Sigma 4764). These amounts varied from125ng to 4000ng per well. 40µl of sample (or standard) was mixed with 160µl of the fluorescentdye in a white-bottom 96-well-plate (Falcon), incubated for 20 minutes in the dark, and absorbtionwas measured in a Perkin Elmer 7000 bioassay-reader at an emission wavelength of 465nm andand excitation wavelength of 360nm. All measurements were done in duplicates.Results gave the amount of DNA (ng) per scaffold. In literature it is found that each chondrocytecontains 7.7pg DNA [25]. This amount was used to convert the weight into amount of cells.

2.5.3 GAG-assay

The amount of GAG was evaluated using a DMMB-solution (0.04mM 1,9-Dimethyl-methyleneblue (Sigma 341088), 40mM glycine(Fluka 50056), 40mM NaCl (Fluka 71380)) as described byFarndale et al. [11]. The amount of GAG’s was measured against a standard curve consisting of aknown amount of chondroitin 4-sulfate (Fluka 27042). These amounts vary from 0.078µg to 2.5µgper well. 20µl of sample was mixed with 200µl of the DMMB-solution in a 96-well-plate (costar).The plate was measured immediately in a Perkin Elmer 7000 bioassay-reader at an absorbanceof 535nm. All measurements were done in duplicates.

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2.5.4 OHP-assay

To 200µl of the digested residue 200µl 12M HCl was added and this was incubated for 12 hoursat 110◦C. The sample was neutralized with 10M NaCl (1.2 times the amount of HCl volume). A1:1 charcoal-resin mixture (Fluka 5110 and BioRad 143-7425) was added. The colorless solutionthat remained was removed (150-200µl) and used for the assay. The amount of hydroxypro-line (OHP) was evaluated using a Chloramine T-solution (0.177g Chloramine T (RiedeldeHaen31224), 10ml MQ-water, 15ml 2-methoxyethanol (BDH 2919865) and 25ml citrate buffer) and aDABA-solution (2.5g DABA (4-Dimethylamino-benzylaldehyd, Fluka 39070), 12.5ml HClO4 (70%,Fluka 77230), 12.5ml MQ-water and 25ml n-propanol (Fluka 82093)). The amount of OHP wasmeasured against a standard curve consisting of a known amount of L-4-hydroxy-proline (Fluka56250). These amounts vary from 0.156µg to 5µg per well. To 200µl of the sample first 200µlsaturated NaCl was added, followed by 400µl Chloramin T solution which was incubated for 4minutes at room temperature. Next 400µl DABA-solution was added and now an incubation pe-riod of 12 minutes at 65◦C was used. After cooling down to room temperature 250µl of thismixture was measured in a Perkin Elmer 7000 bioassay-reader at an absorbance of 560nm. Allmeasurements were done in duplicates.

2.6 Viability assay

A quarter of the scaffold was put in 1ml fluorescent dye (8ml plain DMEM is supplemented with2µl calcein AM and 4µl ethidium homodimer (both from Molecular Probes L-3224)). Calcein AMbecomes green fluorescent after conversion by cytoplasmic esterase (living cells) and ehtidiumhomodimer fluoresces red when bound to DNA (only possible in dead cells). The scaffold wasincubated for 30 minutes at 37◦C and washed 10 minutes at 37◦C in plain DMEM.The scaffolds were evaluated on a laser scanning confocal microscope (Zeiss LSM510) within 1hour after staining, to prevent photo-bleaching. The original center of the scaffold was scanned ina 20µmstack from 50-70µmdeep, to prevent counting dead cells due to cutting.

2.7 Histology

A whole alginate bead and a quarter of each scaffold were used for histology. Different stainswere performed on the sections cut from the samples. Matrix production by the chondrons inan alginate bead was visualized with an alcian blue (GAG) and a Hematoxylin/ Fast Green stain(collagen) stain. The scaffolds were stained with a toluidine blue (overall matrix production) and aHematoxylin/ Safranin O and Fast green stain (GAG & collagen). Immunohistology was performedfor aggrecan, link protein, collagen II and collagen I. All sections were analyzed on a bright fieldmicroscope (Axioplan) with different magnifications.

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2.7.1 Preparation of the samplesBefore histology could be performed the scaffolds were fixed in 25ml 100% methanol for at least24 hours to replace all the water in the sample with methanol. The alginate beads were fixedin 25ml 100% methanol containing 1.5% CaCl2. The samples were put in PBS containing 5%sucrose (scaffolds) or dH2O containing 5% sucrose and 1.5% CaCl2 (alginate beads), at least12 hours before cutting, to remove all methanol. The samples were cut on a cryo-tome (MicromHM560MV) with vacuum-system at a specimen temperature of −22◦C and a blade temperatureof −27◦C. The samples were embedded in cryo-compound (Jung, tissue freezing compoundTM)before cutting to apply stability during cutting. The sections from both the alginate beads and thePUR-scaffolds were 12µmthick and cut from the original center of the sample. The sections wereplaced on a positively charged object-glass and stored for at least 24 hours at -20◦C to stimulateattachment to the object-glass.

2.7.2 Alcian blue stainThe slides were washed 3 minutes in 3% acetic acid in 0.1M CaCl2 (Fluka 21075) and werestained for 30 minutes in an alcian blue solution (1 gram Alcian blue 8GX, 0.08M HCl and 0.1MCaCl2 in 250ml MQ-water). The sections were rinsed 10 times in 0.1M CaCl2, followed by 10 dipsin 50 mM CaCl2. The slides were covered with use of hydromount (National Diagnostics) whichwas supplemented with an additional 5mM CaCl2.

2.7.3 Hematoxylin/ Safranin O/ Fast Green stainThe slides were washed in dH2O and stained 15 minutes in Weigerts Hematoxylin (Mikroskopie115973). The differentiation step was performed with 10 dips in acid alcohol, followed by blueingfor 10 minutes in lukewarm tap water (fresh water every couple of minutes). The slides werestained with a 0.02% Fast Green (Fluka 44715) solution in 0.1% Acetic acid (Fluka 45731)) for 5minutes, followed by 30 seconds in a 1% acetic acid solution. Counterstain with 0.1% SafraninO (Schmid GMBH&Co 1B46 3) for 5 minutes. Water was removed from the slides by a seriesof alcohol-solutions; 10 minutes in each concentration (70%, 80%, 96% and 100%) with a freshbatch after 5 minutes. Finally the slides were put in xylene (also 10 minutes and 2 batches) andcover-slipped with Eukitt mount.In case only a Hematoxylin/ Fast Green stain was performed, the counterstain-step with SafraninO was left out and the rest of the procedure was kept the same.

2.7.4 Toluidine blue stainSamples were washed in dH2O and stained for 4 minutes in a filtered toluidine blue solution (3.0gsodiumtetrabrate (Fluka 71998) and 3.0g toluidine blue (Fluka 89640) in 300ml MQ-water). Slideswere rinsed in dH2O again. If the stain was to severe, slides were kept longer in dH2O, if the stainwas to light the slides were put longer in toluidine solution. Sections were dried by blotting themand leaving them 15 minutes for air dry. Samples were put in pure xylene for 2 minutes andtransferred into a new xylene batch for another 5 minutes. Slides were coverslipped with Eukittmount.

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2.7.5 Immunohistochemistry

Immunohistochemistry tests the presence of specific matrix components with the help of anti-bodies. These immunostains have been performed with help of an immunostain-machine. Thismachine performs the complete immunorun after it was properly programmed and all the chemi-cals were loaded in the right concentration and amount. The immunohistochemical staining wasbased on the affinity of avadin D (Vector laboratories R©) to biotin.The sections were analyzed for link-protein, aggrecan and collagen II (all from Development stud-ies hybridoma bank, Univeristy of Iowa) and for collagen I (Sigma). First a circle was drawnaround the sections with a DAKO-pen, to make sure the liquids stayed on the section for thespecified period. Sections were rehydrated 5 minutes with PBS+Tween and washed three times5 minutes with the same solution. The first enzym treatment was done for 30 minutes at 37◦C.Sections were again washed three times five minutes with PBS+Tween. To prevent unspecificattachment of the antibodies, horse serum (species in which the secondary antibody was raised)was used for blocking. The horse serum was incubated for 60 minutes at room temperature. Nextthe primary antibody was added for 30 minutes at room temperature (aggrecan: 1C6 with chon-droitinase AC diluted 1:5, link protein: 8A4 with chondroitinase AC diluted 1:5, collagen II: CIICIwith HC diluted 1:6, collagen I: colI with HC diluted 1:2000) and washed three times five minuteswith PBS+Tween. The secondary antibody was incubated for 30 minutes at room temperature(Anti-mouse biot for all sections, diluted 1:200). Samples were washed three times five minuteswith PBS+Tween. ABC-complex (1ml PBS + 20µl ABC-A + 20µl ABC-B, store at 4◦C and pre-pare at least 30 minutes before use, Vector Laboratories) was applied for 30 minutes at roomtemperature and again the sections were washed three times five minutes with PBS+Tween. 3,3’-Diaminobenzidine monomer (DAB) was added as a substrate and incubated for 4 minutes in thedark at room temperature. The enzyme on the antibody oxidized the substrate forming a brownishdye deposit, which was observed under a normal bright field microscope.In case of the immunorun for aggrecan and link protein the sections first needed to be reducedand alkylated. This reduction was performed with help of 10mM DTT-solution (1,4-dithiothreitol,Sigma D0632) in a buffer consisting of 50mM Tris (Sigma T1503) en 200mM NaCl (Fluka 71379).Sections were placed in this solution for 2 hours at 37◦C. The alkylation process was performedwith a 40mM Iodoacetamide (Sigma I6125) solution in PBS for 1 hour at 37◦C.

2.8 Statistical evaluation

Data obtained with the assays were quantitative data, so statistical analysis could be performed.Data are presented as the mean ± st.dev. in bar-plots. Two-way analysis of variance (2-way-ANOVA) with bonferroni post-hoc testing was performed with SPSS-software c© and significancelevels p<0.05. Differences between cell-type seeded and culture period were investigated.

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Results

3.1 General observationsChondrocytes were harvested smoothly without any chances for contaminations. The seedingof the alginate beads worked fine as well, only a little more cells per bead were obtained asexpected. Still the cells developed nice during culture (observed with a light microscope). Theseeding of the PUR-scaffolds was in both cases critical (as results will show). The fibrin did gelatewithin the hour incubating, but after 2 days of culture some of fibrin came back out of the scaffold.This also happened during all the trials, so it seemed not to cause any relevant trouble. Cultureof the samples showed no curiosities, no contaminations were observed and medium-changeswere punctual.Samples were properly fixed for at least 24 hours and the cutting demanded some effort, but inthe end the cuts showed nice results.The DNA- and GAG-assay worked without any problems, but the OHP-assay did not produce anyusable data. Most likely one of the chemicals got expired and made the reaction fail. The toluidineblue stain showed dark spots immediately after staining, but these spots could be washed awayin dH2O. The hematoxylin/ fast green/ safranin O stain had technical difficulties, but only afteruse it could be observed if it worked properly or not.

3.2 Assay resultsA significant increase in the amount of cells is found between 2 and 21 days of culture (chondro-cytes seeded in PUR, p=0.01). An increase in total cell number is also present when chondronsare seeded in PUR, but this increase is not statistically significant. This indicates that in bothexperiments the cells are active and proliferating.After 21 days of culture a significant difference (p=0.01) between chondrocyte- and chondron-seeded constructs is found for the cell-type seeded. It should be noted that the chondrons areonly cultured in the scaffold for 11 days.Interestingly also an interaction between culture period and cell-type is found with two-way-ANOVA.

A significant increase in GAG/DNA is found between 2 and 21 days of culture (chondrocytesseeded in PUR, p=0.05). The amount of GAG/DNA also increases in the scaffolds seeded withchondrons, but this increase is not significant. Again this indicates that the cells are active andproduce (more) matrix over time.

17

18

0

1

2

3

4

5

6

7

8

9

10x 106

Cel

ls p

er s

caffo

ld

Cells/ scaffold

2 days 11 days +10d alginate

21 days 2 days +10d alginate

** **

single cells vs. artificial chondrons

scaffolds seeded with single cells scaffolds seeded with artificial chondrons

0

1

2

3

4

5

6

GAG/ DNA per scaffold

GA

G/ D

NA

*

2 days 21 days 2 days +10d alginate

11 days +10d alginate

single cells vs. artificial chondrons

scaffolds seeded with single cells scaffolds seeded with artificial chondrons

Figure 3.1:Results of the DNA-assay and the GAG-assay presented as the total amount of cells per scaffold (left)and the GAG produced per DNA (right) (* significant for p=0.05, ** significant for p=0.01)

The amount of GAG/DNA is not significantly different two days after seeding, but a lower mean isfound for scaffolds seeded with chondrocytes. After 21 days the GAG/DNA levels for both experi-ments are not significantly different.

Just before seeding the viability of the cells was 95-98%, so experiments started with viable cells.Figure 3.2 shows results for the viability assay (Green = viable, red = dead). Hardly any cells arefound in the center of the scaffolds. Near the edges of the scaffolds more cells can be found. Mostof these cells appear to be dead (60-75%).

2 days cultured in PUR

after 10 days in alginate 21 days cultured in PUR

11 days cultured in PUR

after 10 days in alginate

Viability assay

Ce

nte

rSid

e

100,00µm100,00µm

100,00µm100,00µm100,00µm

100,00µm

Figure 3.2:Pictures obtained from scaffolds stained for viability. Green cells are viable, red cells are dead. Shownimages are projections of stacks from 20µmthick, taken from 50-70 µm into the scaffold.

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3.3 Histology results

Figure 3.4 (see in two pages) shows that 2 days after seeding chondrocytes, no matrix-productionis found either at the edge nor in the center of the scaffold. Not many cells have reached the cen-ter of the scaffold. Immunohistochemistry for these samples shows no positive results for eitheraggrecan, link protein, collagen II or collagen I (results not presented)

Figure 3.5 shows the results for scaffolds seeded with chondrons and cultured for 2 days. Againthe cells have not reached the center of the scaffold. A fibrin clot attached to the scaffold canbe observed in Figure 3.5a. Within this clot the chondrons show a homogenous distribution andsome matrix production (Figure 3.5e) The hematoxylin/ safranin O/ fast green stain, has a strongbackground stain so it is hard to determine if the cells show a positive stain for the safranin O(rectangles in figure 3.5g). It is not possible to say anything about the matrix produced in thealginate bead, but Figure 3.3 (below) shows an alginate bead after 10 days of culture stained withalcian blue, hematoxylin/ eosin and hematoxylin/ fast green. All these stains show positive cells(rectangles).Immunohistochemistry for the scaffolds shows that the chondrons produced some collagen II anda little collagen I (Figure 3.6). No aggrecan and link protein are observed.

Alginate beads cultured for 10 days with chondrocytes

Hematoxylin- Fast Green Alcian Blue Hematoxylin- Eosin

Figure 3.3:Results of a hematoxylin/ fast green and a alcian blue stain after 10 days of culture in a alginate bead

20

Figures 3.7 and 3.8 show the results for scaffolds seeded with chondrocytes and cultured for 21days. The toluidine blue stain shows a lot of matrix production along the edge of the scaffoldand also matrix production around the few cells in the center of the scaffold (rectangles in Figure3.7d+e). These findings are again not supported by the hematoxylin/ safranin O/ fast green stain(rectangles in Figure 3.7f+g).Immunohistochemistry on these sample shows no positive results for the aggrecan stain. Linkprotein does show clear positive result in some regions (rectangles in Figure 3.8). Collagen II isproduced a lot within the scaffold and collagen I is only produced at the outermost layer of thescaffold.

Figures 3.9 and 3.10 show the results for the scaffolds seeded with chondrons and cultured for11 days in PUR. Again little cells are observed in the center of the scaffold, but the few cells didproduce matrix (Figure 3.9e+g). Around the edge of the scaffold matrix was produced as is con-firmed by the toluidine blue stain (Figure 3.9d). Again the hematoxylin/ safranin O/ fast green stain(Figure 3.9f) did not show the expected results. Most likely this is due to an technical difficulty.The samples do not show any positive results for aggrecan. Some cells did produce matrix thatcontains link protein (rectangles in Figure 3.10). Collagen II and collagen I are produced less thanin the case of the single cells, but both are present at the same places as in the single cell seededconstructs.

Table 3.1 presents an overview of the stain-intensity observed for each stain within the 4 groups.

Table 3.1: Summary of the stain-intensity for the 4 experiments

Toluidine Hematoxylin/ Linkblue saf. O/ f. green Aggrecan protein Collagen II Collagen I

2 day cells -- -- -- -- -- --2 day chondrons + -+ -- -- + -+21 day cells ++ + - -+ ++ +21 day chondrons + -+ -- -+ + +

21

Single cells cultured 2 days in PUR

Toluidine blue

Hematoxylin- Safranin O- Fast Green

A

B C

GF

D E

Figure 3.4:Results of a toluidine blue and a hematoxylin/ safranin O/ fast green stain after 2 days of culture ofchondrocytes in a PUR-scaffold

22

Toluidine blueChondrons cultured 2 days in PUR

A

B C

D E

F G

Hematoxylin- Safranin O - Fast Green

Figure 3.5:Results of a toluidine blue and a hematoxylin/ safranin O/ fast green stain after 2 days of culture ofchondrons in a PUR-scaffold

23

Aggrecan

Control

Collagen II Collagen I

Link

Control

Chondrons cultured 2 days in PUR

Immunohistochemistry

Figure 3.6:Results of immuno-stains for aggrecan, link protein, collagen II & collagen I after 2 days of culture ofchondrons in a PUR-scaffold

24

A

B C

F G

Hematoxylin- Safranin O- Fast Green

Single cells cultured 21 days in PUR

Toluidine blue

D E

Figure 3.7:Results of a toluidine blue and a hematoxylin/ safranin O/ fast green stain after 21 days of culture ofchondrocytes in a PUR-scaffold

25

Aggrecan Link Protein

Collagen II Collagen I

Control

Single cells cultured 21 days in PUR

Immunohistochemistry

Figure 3.8:Results of immuno-stains for aggrecan, link protein, collagen II & collagen I after 21 days of culture ofchondrocytes in a PUR-scaffold

26

Chondrons cultured 11 days in PUR

Toluidine blue

Hematoxylin- Safranin O- Fast Green

A

B C

GF

D E

Figure 3.9:Results of a toluidine blue and a hematoxylin/ safranin O/ fast green stain after 11 days of culture ofchondrons in a PUR-scaffold

27

Aggrecan Link Protein

Collagen II Collagen I

Control

ImmunohistochemistryChondrons cultured 11 days in PUR

Figure 3.10:Results of immuno-stains for aggrecan, link protein, collagen II & collagen I after 11 days of cultureof chondrons in a PUR-scaffold

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Discussion & Conclusion

Natural chondrons cultured in pellet or agarose constructs produce more matrix over time thanchondrocytes [17][24]. In pellet culture chondrons show a better cell separation and a cell-densityapproaching the cell-density of natural cartilage indicating a more homogenous matrix [26].Results obtained with this study showed that the amount of DNA was significant higher with chon-drocytes and histology shows that much more matrix is produced by chondrocytes, but the amountof GAG/DNA was the same for both chondrons and chondrocytes. This contradiction with litera-ture is most likely due to the fact that in this study all chondrocytes were cultured the same periodfrom harvesting and therefore not for the same period inside the PUR-scaffold.

Histological results showed that the cells did not migrate into the scaffold during the seedingprocedure and almost all cells ended up near the edge of the scaffold or in a fibrin clot attached tothe scaffold. This is a major problem, since it limits the usability of all results. Results will still beinterpreted, since 2 days after seeding the scaffold both chondrocytes and chondrons show thesame amount of cells present in a scaffold. This indicates that the seeding procedure caused thesame error in both groups.

During seeding no remarkable observations were made. The fibrin seemed to gelate properlyand was distributed all around the scaffolds. A layer of fibrin was found on top of the scaffoldsat the first media-change, as usually. Previous experiments with PUR-scaffolds showed homoge-nous distribution of cells and fibrin throughout the scaffolds [16][28][51].In order to prevent the chondrons from blocking the canals during seeding the pore-size of thescaffolds used in this report ranged from 400-600µmwhile previous studies used scaffolds withpore-sizes ranging 200-400µm. This difference was not thought to cause any problems. However,hardly any cells are found in the center of the scaffold. Therefore, it is likely that the fibrin/cell-mixture did not enter the scaffold. There is no explanation for this discrepancy with former reports.

As mentioned before, the total amount of chondrons in a PUR-scaffold is not significantly in-creased within the culture-period of 11 days. The amount of GAG’s neither increases significantlyover time. According to histology the amount of GAG’s did increase over time, because link-protein showed positive results after 11 days, but not after 2 days. However, aggrecan does notshow positive in both cases. Since link-protein does show positive results, this indicates that theimmunostain for aggrecan did not work properly, because link protein is part of the aggrecan-aggregate [35].

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30

The amount of collagens can not be represented quantitatively, since the OHP-assay did not resultin any data. Most likely one of the chemicals lost its function and since there was no time left, itcould not be performed again. Immuno-histology for collagens shows that chondrocytes producemuch more collagen II in 21 days, than artificial cultured chondrons do. In both cases collagen Ican only be found near the edges of the sample, indicating that in both cases the samples stayedchondrogenic. Taking into account that less cells are observed after 11 days of culturing chon-drons in PUR, it is not obvious anymore that the chondrocytes did produce more collagens overtime and information of the OHP-assay is needed to draw any conclusions.

The viability assay shows that there are not many cells present in the center of the scaffold.Around the edge of the scaffold cells are found, but many cells (50-60%) appear to be dead. Thisspecific viability assay has been used a lot in house and has also been used for the scaffoldswhen seeded with chondrocytes [16]. Since the cells in the scaffold did produce matrix and thuswere viable, the high amount of dead cells was unexpected. However, it is still possible that thecells died during procedures before staining or even due to the staining.In literature it is found that both chondrocytes and chondrons maintain a high viability during 2weeks of culture (75-80%) in a alginate bead [30]. There is no reason to assume this to be differ-ent this time. Right after harvesting the chondrocytes showed a viability of 95-98%, but it can notbe assumed that the viability is the same in an alginate bead or a PUR-scaffold.

In conclusion not many conclusions can be drawn from the results obtained in this study. Becausecells were not dispersed in the tissue, but remained positioned at the boundaries, homogenousmatrix-distribution cannot be expected. Also the viability assay did not provide any reliable results.

Recommendations

A follow-up experiment is necessary to state anything on the hypothesis set in the introduction.Trials should be performed to recover possible problems with the scaffold or the fibrin during seed-ing. Scaffolds with the original pore-sizes (200-400µm) could be compared to scaffolds used inthis report (pore-size 400-600µm). Also different concentrations of fibrin and thrombin could betested in order to improve the entire seeding-protocol.

Once this problem is solved a new set of experiments should be scheduled. This set-up shouldbe the same as the one presented in this report, only one group with chondrons extracted fromthe cartilage and seeded immediately in a PUR-scaffold should be added. To compare the dif-ference between cells and chondrons that were cultured for the same period. Furthermore anextra monitor-point should be added from both the extracted chondron-group and the chondrocytegroup. After 11 days of culture inside the PUR-scaffold, scaffolds should be fixed and analyzed aswell. To compare the results from the artificial chondrons to the results from extracted chondronsand single chondrocytes.

With this new experiment it is also necessary to have all (biochemical) assays and histologicalstains working properly. This means that for the OHP-assay new chemicals should be orderedand the assay should be tested. The viability assay should be performed on scaffolds that docontain cells in the center to validate the use of the assay with PUR-scaffolds. Finally, the anti-body for aggrecan should be tested for specificity to see if this was the problem in the aggrecan-immunostain.

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Bibliography

[1] Sudha Agarwal, Robert Gassner, Nicholas P. Piesco, and Sudhaker R. Ganta. Tissue en-gineering and biodegradable equivalents - Scientific and clinical applications, volume 1,chapter 7 - ”Biodegradable urethanes for biomedical applications”, pages 123–144. Mar-cel Dekker Inc., 270 Madison Avenue, New York, NY 10016, 2002.

[2] Charles W. Archer and Philippa Francis-West. The chondrocyte. The international journal ofbiochemistry and cell biology, 35(4):401–404, April 2003.

[3] A. Benninghoff. Form und bau der gelenkknorpel in ihren beziehungen zur funktion. zweiteteil. der aufbau des gelenkknorpels in seinen beziehungen zur funktion. Zeitschrift Zell-forschung, 2:783–862, 1925.

[4] Lawrence J. Bonassar. Methods of tissue engineering, chapter 93 - ”Cartilage reconstruc-tion”, pages 1027–1039. Academic Press, 2002.

[5] N.D. Broom and D.B. Myers. A study of the structural response of wet hyaline cartilage tovarious loading situations. Connective tissue research, 7(4):227–237, 1980.

[6] Qing Cai, Jian Yang, Jianzhong Bei, and Shenguo Wang. A novel porous cells scaf-fold made of polylactide-dextran blend by combining phase-separation and particle-leachingtechniques. Biomaterials, 23(23):4483–4492, December 2002.

[7] Natasha D. Case, Angel O. Duty, Anthony Ratcliffe, Ralph Muller, and Robert E. Guldberg.Bone formation on tissue-engineered cartilage constructs in vivo: Effects of chondrocyteviability and mechanical loading. Tissue Engineering, 9(4):587–596, August 2003.

[8] John T. Connelly, Eric J. Vanderploeg, and Marc E. Levenston. The influence of cyclic ten-sion amplitude on chondrocyte matrix synthesis: Experimental and finite element analysis.Biorheology, 41(3-4):377–387, 2004.

[9] Charles J. Doillon. Methods of tissue engineering, chapter 46 - ”Modification of natural poly-mers: Fibrinogen - fibrin”, pages 555–563. Academic Press, 32 Jamestown road, LondonNW1 7BY, UK, 2002.

[10] Daniela Eyrich, Ferdinand Brandl, Bernhard Appel, Hinrich Wiese, Gerhard Maier, Magda-lene Wenzel, Rainer Staudenmaier, Achim Goepferich, and Torsten Blunk. Long-term stablefibrin geld for cartilage engineering. Biomaterials, 28(1):55–65, January 2007.

33

34

[11] Richard W. Farndale, David J. Buttle, and Alan J. Barrett. Improved quantitation and discrim-ination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica etBiophysica Acta (BBA) - General Subjects, 883(2):173–177, September 1986.

[12] Lisa E. Freed, Gordana Vunjak-Novakovic, Robert J. Biron, Dana B. Eagles, Daniel C.Lesnoy, Sandra K. Barlow, and Robert Langer. Biodegradable polymer scaffolds for tissueengineering. Nature Bio-Technology, 12:689–693, 1994.

[13] A. Gigout, M. Jolicoeur, and M.D. Buschmann. Low calcium levels in serum-free mediamaintain chondrocyte phenotype in monolayern culture and reduce chondrocyte aggregationin suspension culture. Osteoarthritis & Cartilage, 13(11):1012–1024, November 2005.

[14] Katarzyna Gorna and Sylwester Gogolewski. Synthetic bioresorbable polymers for implants,chapter Novel biodegradable polyurethanes for medical applications, pages 39–57. 1396.American society for testing and materials, 2000.

[15] Katarzyna Gorna and Sylwester Gogolewski. In vitro degradation of novel medicalbiodegradable aliphatic polyurethanes based on ε-caprolactone and pluronics with varioushydrophilicities. Polymer Degradation and Stability, 75(1):113–122, 2002.

[16] Sibylle Grad, Laszlo Kupcsik, Katarzyna Gorna, Sylwester Gogolewski, and Mauro Alini. Theuse of biodegradable polyurethane scaffolds for cartilage tissue engineering: potential andlimitations. Biomaterials, 24(28):5163–5171, December 2003.

[17] Ronald D. Graff, Eduardo R. Lazarowski, Albert J. Banes, and Greta M. Lee. Atp re-lease by mechanically loaded porcine chondrons in pelllet culture. Arthritis and rheumatism,43(7):1571–1579, July 2000.

[18] Gregor T. Grant, Edwin R. Morris, David A. Rees, Peter J.C. Smith, and Davis Thom. Biolog-ical interactions between polysaccharides and divalent cations: The egg-box model. FEBSLetters, 32(1):195–198, May 1973.

[19] Zbigniew Gugula and Sylwester Gogolewski. In vitro growth and activity of primary chon-drocytes in a resorbable polylactide three-dimensional scaffold. volume 43rd ORS Anualmeeting, pages 183–191, San Fransisco, California, June 1999. AO/ASIF research institute,polymer research, John Wiley and sons inc.

[20] Hans J. Hauselmann, Russell J. Fernandes, Su S. Mok, Thomas M. Schmid, Joel A. Block,Margaret B. Aydelotte, Klaus E. Kuettner, and Eugene J.-M.A. Thonar. Phenotypic stabilityof bovine articular chondrocytes after long-term culture in alginate beads. Journal of cellscience, 107(1):17–27, January 1994.

[21] Yi Hong, Haiqing Song, Yihong Gong, Zhengwei Mao, Changyou Gao, and Jiacong Shen.Covalently crosslinked chitosan hydrogel: Properties of in vitro degradation and chondrocyteencapsulation. Acta biomaterialia, 3(1):23–31, January 2007.

35

[22] Dietmar W. Hutmacher. Scaffolds in tissue engineering bone and cartilage. Biomaterials,21(24):2529–2543, December 2000.

[23] A.K. Jeffery. Articular cartilage and the orthopaedic surgeon. part i: Structure and function.Current Orthopaedics, 8(1):38–44, January 1994.

[24] Terri-Ann N. Kelly, Christopher C.-B. Wang, RObert L. Mauck, Gerard A. Ateshian, andClark T. Hung. Role of cell-associated matrix in the development of free-swelling and dy-namically loaded chondrocyte-seeded agarose gels. Bioreheology, 41(3-4):223–237, May2004.

[25] Young-Jo Kim, Robert L.Y. Sah, Joe-Yuan H. Doong, and Alan J. Grodzinsky. Fluorometricassay of dna in cartilage explants using hoechst 33258. Analytical biochemistry, 174(1):168–176, October 1988.

[26] Christopher M. Larson, Scott S. Kelley, A. Degene Blackwood, Albert J. Banes, and Greta M.Lee. Retention of native chondorcyte pericelular matrix results in significantly improved ma-trix production. Matrix biology, 21(4):349–359, June 2002.

[27] C.T. Laurencin, A.M.A. Ambrosio, M.D. Borden, and J.A. Cooper Jr. Tissue engineering:Orthopedic applications.

[28] Cynthia R. Lee, Sybille Grad, Katarzyna Gorna, Sylwester Gogolewski, Andreas Goessi,and Mauro Alini. Fibrin-polyurethane composites for articular cartilage tissue engineering: Apreliminary analysis. Tissue engineering, 11(9-10):1562–1573, September 2005.

[29] David A. Lee and Dan L. Bader. Compressive strains at physiological frequencies influ-ence the metabolism of chondrocytes seeded in agaraose. Journal of orthopaedic research,15(2):181–188, March 1997.

[30] Greta M. Lee, C. Anthony Poole, Scott S. Kelly, Jiang Chang, and Bruce Caterson. Isolatedchondrons: a viable alternative for studies of chondrocyte metabolism in vitro. Osteoarthritisand cartilage, 5(4):261–274, July 1997.

[31] H. Janice Lee, Jin-Soo Lee, Thaniussara Chansakul, Christopher Yu, Jennifer H. Elisseeff,and Seungju M. Yu. Collagen mimetic peptide-conjugated photopolymerizable peg hydrogel.Biomaterials, 27(30):5268–5276, October 2006.

[32] A. Maroudas. Balance between swelling pressure and collagen tension in normal and de-generative cartilage. Nature, 260(5554):808–809, April 1976.

[33] Koichi Masuda, Robert L. Sah, Michael J. Hejna, and Eugene J.-M. A. Thonar. A noveltwo-step method for the formation of tissue-engineered cartilage by mature bovine chondro-cytes: The alginate-recovered-chondrocyte (arc) method. Journal of orthopeadic research,21(1):139–148, January 2003.

36

[34] R.L. Mauck, C.C-B. Wang, E.S. Oswald, G.A. Ateshian, and C.T. Hung. The role of cell seed-ing density and nutrient supply for articular cartilage tissue engineering with deformationalloading. Osteoarthritis and cartilage, 11(12):879–890, December 2003.

[35] Van C. Mow, Wie Yong Gu, and Faye Hui Chen. Basic orthopaedic biomechanics andmechano-biology, chapter 5 - ”Structure and function of articular cartilage and meniscus”,pages 181–258. Lippincott Williams & Wilkins, 2005.

[36] C. Anthony Poole. Review - articular cartilage chondrons: form, function and failure. Journalof anatomy, 191(1):1–13, July 1997.

[37] C. Anthony Poole, Michael H. Flint, and Brent W. Beaumont. Chondrons in cartilage: Ultra-structural analysis of the pericellular microenvironment in adult human articular cartilages.Journal of orthopaedic research, 5(4):509–522, 1987.

[38] C. Anthony Poole, Michael H. Flint, and Brent W. Beaumont. Chondrons extracted from ca-nine tibial cartilage: Preliminary report on their isolation and structure. Journal of orthopeadicresearch, 6(3):408–419, 1988.

[39] C. Anthony Poole, Akira Matsuoka, and Jennifer R. Schofield. Chondrons from articularcartilage: Iii morphologic changes in the cellular micorenvironment of chondrons isolatedfrom osteoarthritic cartilage. Arthritis and rheumatism, 34(1):22–35, January 1991.

[40] Mark A. Randolph, Kristi Anseth, and Michael J. Yeremchuck. Tissue engineering of carti-lage. Clinical Plastic Surgery, 30(4):519–537, October 2003.

[41] N. Schneider, J.-P Lejeune, C. Deby, G.P. Deby-Dupont, and D. Serteyn. Viability of equinearticular chondrocytes in alginate beads exposed to different oxygen tensions. The Veteri-nary Journal, 168(2):167–173, September 2004.

[42] Xin Xin Shao, Dietmar W. HUrmacher, Saey Tuan Ho, James C.H. Goh, and Eng Hin lee.Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondraldefects in rabbits. Biomaterials, 27(7):1071–1080, March 2006. scaffolds in TRE.

[43] Martin Stoddart, Ladina Ettinger, and Hans J. Hauselmann. Generation of a scaffold freecartilage-like implant for a small amount of starting material. Journal of cellular and molecularmedicine, 10(2):480–492, April-June 2006.

[44] J.A. Szirmai. Structure of cartilage in ageing of connective and skeletal tissue. Stockholm:Nordiska Bokhandelns, 1969.

[45] J.M. Taboas, R.D. Maddox, P.H. Krebsbach, and S.J. Hollister. Indirect solid free form fab-rication of local and global porous biomimetic and composite 3d polymer-ceramic scaffolds.Biomaterials, 24(1):181–194, January 2003.

37

[46] Rahul S. Tare, Daniel Howard, Jodie C. Proud, Helmtrud I. Roach, and Tichard O.C. Oreffo.Tissue engineering strategies for cartilage generation - micromass and three dimensinalcultures using human chondrocytes and a continuous cell line. Biochemical and biophysicalresearch communications, 333(2):609–621, July 2005.

[47] Johnna S. Temenoff and Antonios G. Mikos. Review: Tissue engineering for regeneration ofarticular cartilage. Biomaterials, 21(5):431–440, March 2000.

[48] B. Thu, P. Bruheim, T. Espevik, O. Smidsrød, P. Soon-Shiong, and G. Skjak-Bræk. Alginatepolycation capsules: I. interaction between alginate and cation. Biomaterials, 17:1031–1040,1996.

[49] Stephan D. Waldman, Caroline G. Spiteri, Marc D. Grynpas, Robert M. Pilliar, and Rita A.Kandel. Long-term intermitent shear deformation improves the quality of cartilaginous tissueformed in vitro. Journal of orthopaedic research, 21(4):590–596, 2003.

[50] Xuanhui Wang, Shawn P. Grogan, Franz Rieser, Verena Winkelmann, Veronique Maquet,Martine La Berge, and Pierre Mainil-Varlet. Tissue engineering of biphasic cartilage con-structs using various biodegradable scaffolds: an in vitro study. Biomaterials, 25(17):3681–3688, August 2004.

[51] E. Wernike. The influence of loading under confined compression at either 5% or 21%oxygen tension on chondrocyte metabolic activity. Diploma thesis, AO Research Institute &Martin-Luther-Universitaet, 2006.

[52] Hua Yang and James R. Wright Jr. Methods of tissue engineering, chapter 69 - ”Microen-capsulation methods: Alginate”, pages 787–801. Academic Press, 2002.

[53] I. Youn, J.B. Choi, L. Cao, L.A. Setton, and F. Guilak. Zonal variations in the three-dimensional morphology of the chondron measured in situ using confocal microscopy. Os-teoarthritis and cartilage, 14(9):889–897, September 2006.

[54] Z. Zhang, J. Fan, K.G. Becker, R.D. Graff, G.M. Lee, and C.A. Francomano. Comparison ofgene expression profile between human chondrons and chondrocytes: A cdna microarraystudy. Oseoarthritis & Cartilage, 14(5):449–459, May 2006.

[55] http://tollefsen.wustl.edu/projects/coagulation/fig11.gif.

[56] http://w3.ouhsc.edu/histology/Glass%20slides/71 03b.jpg.

[57] http://www.ispcorp.com/products/food/content/brochure/alginates/reaction.html.

[58] http://www.lsbu.ac.uk/water/hyalg.html.