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International Journal of Polymeric Materials andPolymeric Biomaterials

ISSN: 0091-4037 (Print) 1563-535X (Online) Journal homepage: http://www.tandfonline.com/loi/gpom20

A novel strategy for cartilage tissue engineering:Collagenase-loaded cryogel scaffolds in a sheepmodel

Burak Gürer, Cengiz Yılmaz, Ş. Necat Yılmaz, Sertan Çabuk & Nimet Bölgen

To cite this article: Burak Gürer, Cengiz Yılmaz, Ş. Necat Yılmaz, Sertan Çabuk & Nimet Bölgen(2017): A novel strategy for cartilage tissue engineering: Collagenase-loaded cryogel scaffoldsin a sheep model, International Journal of Polymeric Materials and Polymeric Biomaterials, DOI:10.1080/00914037.2017.1327433

To link to this article: https://doi.org/10.1080/00914037.2017.1327433

Accepted author version posted online: 09Jun 2017.Published online: 07 Sep 2017.

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INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS https://doi.org/10.1080/00914037.2017.1327433

A novel strategy for cartilage tissue engineering: Collagenase-loaded cryogel scaffolds in a sheep model Burak Gürera, Cengiz Yılmaza, Ş. Necat Yılmazb, Sertan Çabuka and Nimet Bölgenc

aDepartment of Orthopedics and Traumatology, Medical School, Mersin University, Mersin, Turkey; bDepartment of Histology and Embryology, Medical School, Mersin University, Mersin, Turkey; cChemical Engineering Department, Engineering Faculty, Mersin University, Mersin, Turkey

ABSTRACT In this study, we developed a novel strategy, through which cartilage tissue pieces were placed in a sheep cartilage defect model and covered with a collagenase incorporated cryogel scaffold (in vivo cartilage tissue engineering, IVCTE group). While applying this strategy, the chondrocytes could be isolated inside the body and the treatment could be accomplished in one session. To compare our strategy, to another group, in which we used cultured cells and Chondro-gide, standard matrix-induced autologous chondrocyte implantation (MACI) was applied. Although the MACI applied group demonstrated better healing than IVCTE, the type II collagen synthesis was better in the IVCTE group compared to MACI applied group. Collagenase did not have detrimental effect on surrounding cartilage in IVCTE group. The preliminary results of the novel strategy applied group (IVCTE) were promising.

GRAPHICAL ABSTRACT

ARTICLE HISTORY Received 3 February 2017 Accepted 3 May 2017

KEYWORDS Cartilage; cryogel; in vivo; scaffold; tissue engineering

1. Introduction

Cartilage damages may occur as a result of sports or accident injuries, genetic origin malformations, and some cartilage- related disorders. Cartilage tissue, contrary to the bone tissue which has high regeneration capacity, lacks regenerating itself, since it does not have a vascular network. Large chondral defects require a more special attention. Autologous chondro-cyte implantation (ACI) was described as a solution for large

chondral defects. In this cell-based cartilage repair procedure, a piece of cartilage tissue is harvested arthroscopically from a less load-bearing area of the patient. The cells are isolated by removing the extracellular matrix (ECM) enzymatically. The population of the cells are expanded in vitro by cell culture, for 4–6 weeks. The patient then undergoes a second surgery, in which the cells are implanted on the defected area. This technique has its own disadvantages. In this technique,

none defined

CONTACT Nimet Bölgen nimetbolgen@yahoo.com Chemical Engineering Department, Engineering Faculty, Mersin University, Ciftlikkoy Campus, F Blok, 33343, Yenisehir, Mersin, Turkey. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/gpom. © 2017 Taylor & Francis

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patients undergo two operations and the second one is an open surgery. Fixation of the periosteal graft over the defect is technically difficult and prone to failure. Cells in suspension tend to gather at edges rather than distributing homogenously into the volume of the defect or leak inside of the joint. To overcome most of these advantages, matrix-induced auto-logous chondrocyte implantation (MACI) technique, in which autologous chondrocytes are seeded and cultured on type I and II collagen membranes before implantation, was described in 1999, and as of 2002 was accepted as the second generation of ACI [1,2]. With MACI technique, the possibility of leakage of chondrocytes inside of the joint is avoided. Moreover, the membrane can simply be fixed to the defect by gluing rather than being sutured to the surrounding healthy tissue, avoiding the damage to the adjacent cartilage. Although MACI technique does overcome most disadvantages of the afore-mentioned technique, it still requires two surgeries and it is expensive. Both of these modalities utilize cell culture techniques. The cell culture technique depends on isolating mature chondrocytes from their ECM, forcing them to convert into a proliferative phase. Mature chondrocytes which provide continuity of the ECM, when isolated from their lacunae, and if suitable conditions are present, start to proliferate. The suitable conditions are provided by the incubator. Incubator mimics in vivo conditions such as temperature, humidity, CO2, and O2 concentrations, and the nutrition that are readily available in vivo. The cells are incubated in vitro, which takes several weeks, until there is adequate number of cells to reimplant on the damaged area. Moreover, as the number of cell passage increases, the cell phenotype changes.

To overcome these disadvantages, in this study, we developed a new strategy, in which we incorporated cartilage samples in the collagenase-loaded gelatin cryogel scaffolds and implanted them in a sheep cartilage defect. While applying this strategy, chondrocytes could be isolated inside the body, the body could act as a natural incubator for the cells to grow and form the new cartilage tissue. Moreover, the treatment could be accomplished in one session and cost could be reduced. A 2.5 cm diameter full thickness chondral defect was created in knees of three groups of sheep. To one group, cartilage tissue pieces were placed in the defect and covered with a collagenase incorporated cryogel. To another group, standard MACI was applied. Control group was left untreated. After 15 weeks follow-up, repair of the tissues was compared macroscopically, histomorphometrically, and immunohistochemically.

2. Experimental

2.1. Synthesis and characterization of cryogel scaffolds

The cryogel scaffolds were obtained by the following process with modification of our previous study [3]: gelatin (Merck, Germany) was dissolved in 8 mL of distilled water at 6% (w/v) polymer concentration. Glutaraldehyde (25%, v/v, Merck, Germany) solution at concentration of 6% (v/v) was prepared in 2 mL of distilled water. Both solutions were mixed. The mixture was kept in a syringe template under cryogenic conditions (� 16°C) for 16 h and the interconnected macroporous cryogel was formed at the end of a crosslinking

reaction. The cryogel was rinsed with distilled water under room temperature in three periods to dispose unreacted glutaraldehyde and dried at room temperature.

Chemical analysis of pure gelatin and gelatin cryogel scaffold was performed by Fourier transform infrared (FTIR) spectrometry (Frontier Spectrometer-ATR, Perkin Elmer, USA). FTIR spectra of the samples were obtained in the wavelength range of 450–4000 cm� 1, at a resolution of 4 cm� 1.

Scanning electron microscopy (SEM, Supra 55, Zeiss, Germany) was used to investigate the morphology of the synthesized cryogel scaffolds after coating the samples with platinum and palladium. Average pore diameter and pore wall thickness were determined using SEM images.

The swelling ratio of the cryogels was determined according to a previous method demonstrated by Bölgen et al. [4]. The completely dried samples were weighed (Wd). Dried samples were then immersed in distilled water for a certain time. The excess water from the surface of samples was removed using filter paper and the samples were then weighed again (Ww). The ratio of swelling (SR) was calculated using the following equation:

swelling ratio ¼ ðWw � WdÞ=Wd

2.2. In vivo experiments

The experiment was conducted with the permission of the institutional ethics committee. In all, 14 merino female sheep aging between 1 and 2 were used. The sheep were numbered using ear tags and were grouped as follows: six sheep as in vivo cartilage tissue engineering (IVCTE) group, six sheep as MACI group, and two as control group. The sheep were kept in a private fold with unlimited access to food and water during the study. Minimal number of animals was determined by analyzing power of similar previous studies and considering confidence interval as 95%.

All sheep received subcutaneous injection of 7.5% 10 mL cefquinom 30 min prior to surgery and the antibiotic prophy-laxis was carried on for 3 days. Induction of anesthesia was provided by 0.3 mg kg� 1 intramuscular xylazine followed by 2 mg of ketamine injection. Right knees of all sheep were shaved and medial patellar arthrotomy was done via mid- patellar skin incision. A circle 2.5 cm in diameter (approxi-mately 5 cm2) was drawn on the central trochlear cartilage and a chondral defect was created with the help of scalpel and curette. Cartilage including the calcified layer was removed and depth of the defects reached subchondral bone. The procedure was terminated at this level for the control and MACI groups and the surgical incisions were sutured.

For the IVCTE group, the gelatin cryogel scaffold was sterilized with ethylene oxide. The sterilized cryogel was cut to the shape and size of the chondral defect and soaked into 0.2% type II collagenase solution (Biochrom AG, Berlin, Germany) for 30 min. 140–200 mg of the cartilage tissue obtained from the formation of defect was weighed and diced into 1 mm3 pieces making up 10–12 pieces. Base of the defect was covered with fibrin glue (Tisseel, Baxter, USA) and the cartilage tissue pieces were placed into the defect homogenously (Figure 1a). The defect was covered with the collagenase-containing cryogel (Figure 1b). Patella was relocated and the knee was flexed and

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extended repeatedly to test stability of the cryogel and capsule, subcutaneous tissue, and skin were closed properly.

Chondral tissues (140–200 mg of the tissues) obtained from creation of the defects in the MACI group were diced into 1 mm3 pieces and placed into tissue transfer solution (Dulbecco’s modified Eagle’s medium, DMEM). The tissue pieces were rinsed with sterile phosphate-buffered saline (PBS) and placed into 15 mL Falcon tubes containing DMEM (10% fetal bovine serum, 1% penicillin–streptomycin, 1% amphotericin B, 2.5% L-glutamine (Biochrom AG, Berlin, Germany). They were kept in 0.2% type II collagenase (Biochrom AG, Berlin, Germany) overnight and precipitate from each piece was placed in a separate T25 culture flasks. Culture flasks, containing supplemented DMEM, were incubated at 37°C and 5% CO2. Nutrition was provided by changing the medium every 3–4 days until the cells reached confluence. The cells were detached by trypsin, confirmed viability by trypan blue test, and centrifuged to form a pellet. Pellets were collected and seeded on the rough face of precut 2.5 cm diameter type I/III collagen membrane (Chondro-gide, Geistlich Pharma, Wolhusen, Switzerland). The implant was incubated for another 36–48 h. Approximate number of cells per membrane was 2 � 106. The cell expansion process took approximately 6 weeks. At the end of this time, the MACI group sheep were prepared for the second surgery similar to the first one. Previous incision scar was used and the defect was freshened. Any possible bleeding from the subchondral bone was stopped by adrenaline soaked gauze compression. The prepared implant was glued to the base of the defect with Tisseel (Baxter, IL, USA) fibrin glue. Rough face of the collagen membrane on which the chondrocytes were seeded faced the defect and the smooth face faced the joint (Figure 1c). Stability of the construct was tested through flexion and extension, and the incisions were closed.

All animals received 8 ccs of intramuscular metamizole for postoperative analgesia. To prevent weight bearing, a tennis ball was wired to the operated extremity hoof. The tennis balls were removed 6 weeks after the index operation. At the end of the 15th week (for MACI group, 15 weeks after the second operation), all animals were sacrificed and both of the distal femurs were excised.

2.3. Macroscopic assessment

The defective cartilage area was first assessed macroscopically. A scoring system recommended by Rudert et al. [5] was used (Table 1).

2.4. Histologic evaluation

For histologic evaluation, osteochondral samples of the whole defect area, from the intact cartilage distal and adjac-ent to the defect, and from the trochlear region of the con-trolateral knee were obtained. The samples were placed into 10% neutral formaldehyde containing jars and kept 48 h for fixation. After fixation, the tissues were passed through alcohol, xylol, and paraffin solutions, respectively. Five micrometer thick, 10 homogenously distributed sections were made from the paraffin block of the defect area. One section from each intact cartilage sample was made. Similar set of sections were made for immunohistochemical examin-ation. Sections saved for histomorphometric assessment were stained with hematoxylin eosin and safranin-O fast green and examined under light microscope (Nikon Eclipse 80i, Eclipse 80i, Nikon Corp., Japan) and were photographed digitally (Nikon digital camera, DS-L1, Digital camera DS- L1, Nikon Corp., Japan). Sections were scored according to modified O’Driscoll scoring system and average score of the 10 sections were recorded (Table 2).

2.5. Immunohistochemical staining

The tissue samples were further stained immunohisto-chemically for types I and II collagen. Five micrometer thick

Figure 1. (a) IVCTE surgical technique: placement and fibrin glue application of cartilage pieces, (b) implanted gelatin cryogel scaffold, and (c) implanted MACI. Note: IVCTE, in vivo cartilage tissue engineering; MACI, matrix-induced autologous chondrocyte implantation.

Table 1. Scoring system for macroscopic assessment of cartilage healing [5]. Criteria Score Macroscopic properties

Filling 1 Significantly below adjacent cartilage level 2 Same level with adjacent cartilage, central depression 3 Same level with adjacent cartilage

Color 1 Brown or yellow 2 White 3 Same as adjacent cartilage

Surface 1 Rough 2 Smooth

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sections were placed on adhesive lams. Deparaffinized by keeping at 60°C for 1 h and by passing through xylol three times for 10 min; rehydrated by serial application of decreas-ing concentration of alcohol and placing in distilled water; applied trypsin (pH 7.6) at 37°C (Bio-Optica Milano Spa, Italy) to remove antigen masking which may result from fixation or paraffin embedding; washed three times with PBS for 5 min; incubated 10 min in 12.5% hydrogen peroxide (in distilled water) to eliminate endogenous peroxidase activity; washed with PBS again; and incubated with protein block (Novocastra, Leica Microsystems, Germany) for 8 min to inhibit nonspecific protein binding and background dying. After removing the protein block, the samples were incubated at room temperature overnight with primary antibodies. As primary antibodies type I collagen (anti-type I collagen, 1/300, Abcam, Cambridge, UK) and type II collagen (anti-type II collagen, 1/50, Abcam, Cambridge, UK) were diluted with PBS and 0.5% bovine serum albumin. As secondary antibody biotin bonded polyvalent secondary antibody (Scy Tek Laboratories, Logan, UT, USA) was used by incubating for 10 min. Tissue samples were further incubated with streptavidin peroxidase enzyme reagent for another 10 min, dyed with hematoxylin for con-trast. Negative control samples underwent same protocol dis-carding primary antibody application. All samples were examined under Olympus BX50 (Olympus GmBH, Germany) light microscope and were photographed with digital attach-ment Nikon Coolpix5000 (Nikon Corp., Tokyo, Japan). Staining was graded as 0, none; 1, mild; 2, moderate; and 3, strong.

Average score of the defect area samples were recorded.

2.6. Statistical analysis

Macroscopic assessment scores, histomorphometric scores, and immunohistochemical scores of the three groups were compared using Kruskal–Wallis method and the

Table 2. Modified O’Driscoll scoring system [6,7]. Category Score

1. Nature of predominant tissue Cellular morphology

Hyaline articular cartilage 4 Partially differentiated mesenchyme 2 Fibrous tissue or bone 0

2. Structural properties Surface integrity

Smooth and intact 3 Superficial horizontal lamination 2 Fissures: 25–100% of thickness 1 Significant disruption, including fibrillation 0

Structural integrity Normal 2 Slight disruption, including cysts 1 Significant disruption 0

Thickness 100% of normal surrounding cartilage 2 50–100% of normal cartilage 1 0–50% of normal cartilage 0

Bonding at adjacent cartilage Bonded at both ends 2 Bonded at one end or partially at both 1 Not bonded 0

3. Independence from cellular changes of degeneration Hypocellularity

Normal cellularity 3 Mild hypocellularity 2 Moderate hypocellularity 1 Severe hypocellularity 0

Chondrocyte grouping No grouping 2 Less than 25% of cells 1 25–100% of cells 0

4. Independence from degenerative changes of adjacent cartilage Cellular properties

Normal cellularity, no grouping 3 Normal cellularity, mild grouping 2 Mild or moderate hypocellularity 1 Severe hypocellularity 0

Fibrillation No fibrillation 3 Less than 25% of the thickness of cartilage 2 25–50% of the thickness of cartilage 1 More than 50% of the thickness of cartilage 0

Figure 2. FTIR diagram of pure gelatin and gelatin cryogel. Note: FTIR, Fourier transform infrared.

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differing group was determined by Dunn method. Statistical analysis was accomplished using SPSS (SPSS 16.0, Chicago, IL, USA) software. P values lower than 0.05 were considered significant.

3. Results

3.1. Characterization of cryogel scaffold

The chemical groups of pure gelatin and cryogel scaffolds were determined by FTIR. The chemical crosslinking of gelatin with glutaraldehyde was confirmed by FTIR (Figure 2). Gelatin had an amide I peak (C=O stretching) at 1629 cm� 1 and amide II peak (N–H bending) at 1524 cm� 1, which are distinguishing features of gelatin. The spectrum of crosslinked gelatin cryogel is shown in Figure 2. In addition to the previously mentioned peaks, a strong peak at 1448 cm� 1 was observed in the crosslinked gelatin due to aldimine linkage (CH=N) reaction took place during the crosslinking process [8].

The cryogel scaffold was successfully synthesized. Figure 3a shows the image of the dry cryogel. When the cryogel was immersed in aqueous medium, it absorbed the water and reached its equilibrium swollen form in 15th min (Figure 3b). At the end of 30 min, the swelling ratio of the cryogel was 11.6 � 1.11. The image of the cryogel in the swollen form is shown in Figure 3a.

The pore morphology of the cryogel scaffold was investigated by SEM. Figure 4 demonstrates the SEM image of the cryogels at magnifications of �200 and �1000. As seen

Figure 3. (a) Macroscopic image of dry cryogel, (b) swelling ratio profile of cryogel scaffold, and (c) macroscopic image of swollen cryogel.

Figure 4. SEM image of cryogel scaffold at magnification of (a) �200 and (b) �1000. Note: SEM, scanning electron microscopy.

Table 3. Summary of the results.

Sheep number

Macroscopic score

Histomorphometric score

Immunohistochemical score

Type I collagen

Type II collagen

Control group I 3.0 4.0 2.9 1.2 II 4.0 3.9 3.0 1.0 Group

average 3.5 4.0 3.0 1.1

IVCTE group I 8.0 8.4 2.2 2.8 II 5.0 2.5 2.8 2.1 III 6.0 7.4 2.8 1.9 IV 7.0 9.0 3.0 2.1 V 5.0 7.2 2.6 2.9 VI 6.0 8.6 2.5 2.1 Group

average 6.2 7.2 2.7 2.3

MACI group I 6.0 11.9 2.9 1.5 II 8.0 14.8 2.8 1.3 III 5.0 13.9 2.9 1.2 IV 7.0 15.0 2.9 1.1 V 7.0 11.1 2.4 1.6 VI 6.0 15.9 2.9 1.2 Group

average 6.5 13.8 2.8 1.3

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in Figure 4a and 4b, the cryogel contained homogenously distributed interconnected macropores with average diameter of 322.81 � 86.47 µm. The average pore wall thickness of the cryogel was 3.56 � 1.52 µm.

3.2. Macrosopic, histomorphometric, and immunohistochemical scores

Macroscopic scores, histomorphometric scores, and immuno-histochemical scores of the groups were as demonstrated in Table 3. Macroscopically, IVCTE and MACI groups scored better than the non-treated control group (Figure 5). However, the difference between the groups was not statistically significant.

Histomorphometric scores of the MACI group were significantly better than the IVCTE and the control groups (Figure 6). Although IVCTE scored better, the difference between this group and the control group was not significant.

Amount of type I collagen was similar in all groups. Type II collagen amount, when compared to the other two groups, was

significantly more in the IVCTE group (Figure 7). MACI and control groups contained similar amounts of type II collagen.

Cartilage samples obtained from the periphery of the defect were compared macroscopically and histologically to the controlateral knee cartilage. No apparent difference could be observed in structure, ultrastructure, or thickness.

4. Discussion

The crosslinking of gelatin with glutaraldehyde becomes through the reaction of free amino groups (–NH2) of gelatin with aldehyde (–CHO) groups of glutaraldehyde. The characteristic peak of the aldimine groups occurred at 1450 cm� 1 showed successfully fabrication of crosslinked gelatin cryogel [8]. Glutaraldehydes have an aldehyde group (–CHO) that reacts with the amino group of the lysine residues of gelatin.

Swelling ratio is a crucial parameter in the demonstration of the properties of the cryogels. One of the unique characters of the cryogels is their interconnected macroporosity. When a dry cryogel is soaked in an aqueous media, it absorbs the

Figure 5. Macroscopic results. (a) Control group, (b) IVCTE group, and (c) MACI group. Note: IVCTE, in vivo cartilage tissue engineering; MACI, matrix-induced autologous chondrocyte implantation.

Figure 6. Safranin-O dye at �60 magnification. (a) IVCTE group and (b) MACI group. Note: IVCTE, in vivo cartilage tissue engineering; MACI, matrix-induced autologous chondrocyte implantation.

Figure 7. Type II immunohistochemical dye at �60 magnification. (a) IVCTE group and (b) MACI group. Note: IVCTE, in vivo cartilage tissue engineering; MACI, matrix-induced autologous chondrocyte implantation.

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water, quickly reaching its swollen form because of its inter-connected macroporosity. This property is also very useful when a cryogel is designed as a scaffold for a tissue engineering application. As the cryogel is implanted in the defected side, it would quickly absorb the tissue fluids around and fill in the defect properly. The interconnected macropores also provide an adequate environment that enable cells to integrate, contact, and diffuse cellular nutrients and wastes during new tissue formation [4]. In this study, the cryogel scaffold absorbed very quickly the aqueous medium and reached its final swollen form in 15 min.

Scaffolds for tissue engineering applications should have interconnected porous structure to enable cellular penetration, favorable diffusion of nutrients to cells, and allow diffusion of waste products out of the scaffold [3]. Cryogelation is a rather new technique that has been the subject of studies in the last decade in the tissue engineering field. In this study, the fabricated cryogels had interconnected super macroporosity with average pore diameter of 322.81 � 86.47 µm, which would create an adequate environment for cells to penetrate and form new tissue.

Rabbits are the most commonly used experimental subjects in studies about cartilage healing. Tissue engineering studies require larger defects and abundant amount of cartilage tissue. International Cartilage Research Society recommends goat as cartilage defect model [9]. Similar weight sheep have larger knees than goats and resemble more to human knee size, structure, and healing capacity wise [9,10]. We thus preferred sheep as experimental model. Defects with diameter less than 3 mm tend to heal spontaneously [11,12]. Spontaneous healing limit increases up to 6 mm in larger animals [13]. It is known that defects with diameters less than 6 mm heal spontaneously in sheep [14,15]. The created defects in this study were 25 mm in diameter. Lack of spontaneous healing was confirmed by the control group. ACI and MACI are indicated for defects over 2 cm2 in area [16]. Within the 2–5 cm2 range, osteochondral autogenous transfer (OAT) is an alternative treatment [17]. But over 5 cm2, OAT is not recommended and ACI and MACI remain the only solution options. We thus preferred to make the defect around 5 cm2. Defect can either be placed on distal femoral condyles or trochlear groove. A 25 mm diameter defect does not fit to the distal femoral condyle of sheep and thus we created the defects at the trochlear groove. Trochlear groove provides a flat, wide area and has cartilage thickness close to patella [18].

Healthy cartilage tissue is obtained by creation of the chondral defect. This cartilage was used as donor cartilage tissue instead of gathering from elsewhere. For MACI, to proliferate sufficient amount of chondrocytes, 140–200 mg of healthy cartilage tissue is required [19–23]. In this experiment, to simulate the clinical scenario, this amount of the cartilage tissue was used for both MACI and IVCTE groups. The rest of the tissue was discarded.

In the literature, most studies on rabbits did not utilize any kind of immobilization [5,24–26]. For dogs, either external fixation or plaster cast immobilization was used [27]. Sheep mobilization was usually limited by narrowing down fold size [28,29]. We preferred a previous successful method to limit

bearing weight while allowing mobilization and joint motion. In this method, authors fixed a tennis ball under the operated extremity nail and protected repaired tendons for 6 weeks [30].

Original description of MACI included the use of double- layered type I and III collagen membrane [31]. The rough side of the membrane has a loose structure and provides a suitable surface for the cells to attach, proliferate, and produce matrix. The smooth side of the membrane has a dense structure and while allowing nutrition exchange, prevents cell loss [2]. In this study, we produced the MACI implant as was described originally using the same collagen membrane. In the IVCTE group, a gelatin (denaturated collagen) cryogel was used for similar purposes. The gelatin was crosslinked under cryogenic conditions and had a mechanically stable, interconnected, and macroporous structure which would guide new tissue forma-tion, by providing a favorable environment for cells to reside and interact and by maintaining transport of nutrients and wastes through the interconnected pores of the cryogel. The gelatin cryogel also acted as a three-dimensional release system by holding collagenase within.

The major event which forces mature chondrocytes to turn into proliferative phase chondrocytes is removal of the matrix [16]. In cell culture laboratories, this is accomplished mainly by enzymes like type II collagenase. The enzyme digests collagen and the chondrocytes are suspended from their matrix. If proper conditions are provided cells start to prolifer-ate until they reach a confluence at the flask surface. The proper conditions are readily available in vivo. The major problem is to isolate chondrocytes from their mature matrix. In this study, we incorporated collagenase into the cryogel. If collagenase does not perform the intended activity, one would expect mature cartilage tissue islets within the repair tissue. In the IVCTE group, histologic examination revealed homogenous distribution of cells inside the repair tissue and no mature car-tilage tissue was observed. We thus assume that the collagenase achieved to remove the ECM and isolate chondrocytes. In cell culture techniques, growth factors are used to promote chondrocytes to proliferate and synthesize type II collagen. We did not add these growth factors in order not to experiment more than one variable. The promising aspect of the experi-ment was that chondrocytes could be forced to proliferation phase and could be kept in place to produce matrix proteins (type II collagen). When compared to the MACI group IVCTE group, although showed less proliferation and healing, had significantly more type II collagen synthesis.

The first drawback one can think of is that the addition of collagenase into the cryogel may diffuse into the joint and cause generalized chondral damage. The concentration of collagenase used in this experiment is the standard concentration used for chondrocyte cell culture. Lesser concentrations are unable to degrade cartilage matrix. Even if a release of collagenase occurs into the synovial fluid, the concentration distributed to the synovial space would not be sufficient to act on cartilage. We compared the cartilage adjacent to the defect to the cartilage of the controlateral knee. Macroscopically and histologically, both samples from all subjects were identical. Collagenase’s detrimental effects may appear after a longer time follow-up but at least we may conclude that it does not damage surrounding cartilage in

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the acute setting. A repeated synovial fluid analysis for activity of collagenase is currently being carried out.

Cartilage healing takes time. Follow-up time of experimental studies on cartilage healing vary between 6 and 52 weeks [13,18,28,29,32–34]. Although longer follow-up times are better to evaluate rate and durability of healing, they are not always feasible. A group of authors frequently working on cartilage healing on dogs recommend minimal evaluation time as 15 weeks [27]. Current study was terminated at 15 weeks. Longer follow-up might result in better healing or degeneration and worsening.

5. Conclusion

Lack of matrix producing cells in the defected articular carti-lage is considered to be a major cause of poor cartilage repair. Current methods for cartilage restoration typically involve harvesting osteochondral tissue from a non-loaded chondral surface followed by transplantation into the defect site or in situ induction of new cartilage. The methods often suffer from poor integration at the peripheral interface between native and transplanted cartilage or lack of matrix producing cells in the interface area. Bos et al. [35] investigated whether specific enzymatic digestion of the ECM in articular cartilage lesion edges could induce integrative cartilage regeneration via a mechanism of local increase in the number of vital chondro-cytes in cartilage wound edges. They demonstrated that treatment of articular cartilage explants with collagenase could result in a significant increase in chondrocyte density [35]. In another invention, collagenase was applied transiently to digest collagen at the surfaces of the defect, thereby mobilizing the chondrocytes [36]. The cells could then migrate into the defect where they could proliferate and synthesize new ECM. These studies demonstrate strategies to enhance cell release from the cartilage by administration of collagenase. Holloway et al. [37] shortly demonstrated their approach as a conference abstract about treatment of meniscal tear. They prepared elec-trospun poly(e-caprolactone)/poly(ethylene oxide) nanofibrous scaffolds to deliver collagenase in combination with a chemo-kine for enhancing cell mobility and recruitment to overcome limited physical hurdle to cell migration and proliferation due to the high density of ECM in the mature meniscus [36]. To the best of our knowledge, we demonstrated here, the first time in the literature, the novel strategy through which cartilage tissue pieces were placed in a sheep cartilage defect model and covered with a collagenase incorporated cryogel scaffold to isolate chondrocytes inside the body in one session.

As a conclusion, in this study, IVCTE experiment resulted in less healing response compared to MACI. But the chondro-cytes could be isolated and forced to proliferative phase, and they synthesized type II collagen better than the MACI group. Detrimental effect of the collagenase could not be detected to the intact parts of the cartilage. We believe low cost and one session application are major advantages and preliminary results are promising.

Conflict of interest The authors have declared that there is no conflict of interest.

Funding

This study was supported by the Scientific Research Projects Unit of Mersin University (Grant No. BAP-TF CTB (BG) 2010-4 TU).

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