Biomed Tech 2014; 59(5): 375–383

Florian Fensky, Johannes C. Reichert, Andrea Traube, Lars Rackwitz, Sebastian Siebenlist and Ulrich Nöth*

Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels Abstract: Hyaline cartilage displays a limited regenerative potential. Consequently, therapeutic approaches have been developed to treat focal cartilage lesions. Largersized lesions are commonly treated by osteochondral grafting/mosaicplasty, autologous chondrocyte implantation (ACI) or matrix-induced chondrocyte implantation (MACI). As an alternative cell source to chondrocytes, multipotent mesenchymal stem cells (MSCs) are regarded a promising option. We therefore investigated the feasibility of predifferentiating human MSCs incorporated in hydrogels clinically applied for MACI (CaReS®). MSCladen hydrogels were cast and cultured over 10  days in a defined chondrogenic differentiation medium supplemented with TGF-β1. This was followed by an 11-day culture in TGF-β1 free media. After 21 days, considerable contraction of the hydrogels was observed. Histochemistry showed cells of a chondrocyte-like morphology embedded in a proteoglycan-rich extracellular matrix. Real-time polymerase chain reaction (RT-PCR) analysis showed the expression of chondrogenic marker genes, such as collagen type II and aggrecan. In summary, we demonstrate that chondrogenic differentiation of human mesenchymal stem cells embedded in collagen type I hydrogels can be induced under the influence of TGF-β1 over a period of 10 days. *Corresponding author: Prof. Dr. med. Ulrich Nöth, MD, Department of Orthopedics and Accident Surgery, Waldkrankenhaus Protestant Hospital, Stadtrandstraße 555, 13589 Berlin, Germany, Phone: +49 30 3702 1002, Fax: +49 30 3702 2204, E-mail: [email protected] Florian Fensky: Department of Trauma, Hand and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany Johannes C. Reichert, Lars Rackwitz and Ulrich Nöth: Department of Orthopedics and Accident Surgery, Waldkrankenhaus Protestant Hospital, Stadtrandstraße 555, 13589 Berlin, Germany Andrea Traube: Department Laboratory Automation and Biomanufacturing Engineering, Fraunhofer Institute for Manufacturing Engineering and Automation IPA, Nobelstraße 12, 70569 Stuttgart, Germany Sebastian Siebenlist: Department of Traumatology, Technical University of Munich, Ismaningerstraße 22, 81675 Munich, Germany

Keywords: chondrogenic predifferention; collagen hydrogel; human mesenchymal stem cells; matrix-based autologous chondrocyte transplantation. DOI 10.1515/bmt-2013-0076 Received July 15, 2013; accepted April 2, 2014; online first May 6, 2014

Introduction Hyaline articular cartilage has a restricted potential for selfrepair and regeneration. Consequently, focal cartilage defects can subsequently lead to osteoarthritis even in younger patients. For larger-sized osteochondral defects  > 4  cm2 of weight-bearing areas of the knee, techniques established for smaller-sized lesions such as drilling, microfracturing, and transplantation of osteochondral plugs (OATS) provide only insufficient clinical outcomes [9, 25]. As a result, orthopedic research has concentrated on the development of alternative strategies. In 1994, the concept of ACI was introduced [6] with encouraging clinical results [7, 12]. Further developments aiming to address the disadvantages associated with the classic first generation ACI approach gave rise to the tissue engineering based matrix-induced chondrocyte implantation (MACI). MACI relies on the use of biomaterials seeded with chondrocytes as carriers and scaffolds for cell growth to generate new, functional, articular tissue. Over the years, a number of MACI-related products have been commercialized for routine clinical application [2, 13, 34]. One of these, namely the CaReS®-system (Arthro Kinetics plc, Krems, Austria), is composed of autologous chondrocytes seeded into three-dimensional collagen type I hydrogels. Since the system’s introduction in 2003, more than 1000 patients suffering from chondral defects have been treated successfully with this ­technology [2, 3, 23, 32]. The disadvantages associated with the application of autologous cartilage derived cells are chondrocyte

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376      F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels dedifferentiation during in vitro expansion and donor site morbidity. As a result, human mesenchymal stem cells (MSCs) have drawn interest, as these cells show a multilineage differentiation potential [35]. Furthermore, MSCs can easily be harvested from different tissues such as periosteum [26], adipose tissue [38], trabecular bone [27] or bone marrow [30, 36] by minimally invasive techniques. Chondrocytes for MACI using CaReS® are commonly expanded in vitro for 10–12 days. For economic reasons the period of in vitro cell expansion should be as short as possible. TGF-β1 mediated chondrogenic in vitro differentiation of human MSCs, however, is reported to take 21 days [28]. Aiming to apply MSCs for cartilage regeneration using CaReS®, we investigated whether a state of predifferentiation can be achieved within 10 days, which can ultimately lead to chondrogenic differentiation comparable to the degree of differentiation achieved after 21 days of stimulation with TGF-β1.

Materials and methods Isolation and cultivation of MSCs Human mesenchymal stem cells were isolated as described previously and approved by the Institutional Review Board of the University of Würzburg [28, 29]. All tests were carried out at room temperature unless stated otherwise. Briefly, freshly reamed trabecular bone from the femoral head of three patients with no other diseases than osteoarthritis of the hip undergoing total hip arthroplasty was transferred in each case to 50 ml conical tubes (Greiner Bio-One, Frickenhausen) containing standard cell culture medium (SCM). SCM consisted of Dulbecco’s Modified Eagle Medium (DMEM-F12, PAA Laboratories, Linz, Austria) supplemented with 10% fetal bovine serum (FBS, PAA), antibiotics (50 IU penicillin/ml and 50  μg streptomycin/ml, PAA) and ascorbate 2-phosphat (50 μg/ml, Sigma, Steinheim, Germany). A ­ fterwards, the tubes were vortexed to disperse the marrow cells from the bone plugs. After centrifugation to high density pellets, the released cells could be collected. Next, the extracted cells were counted with a hemocytometer and plated at a density of 3 × 106 cells per 175 cm2 tissue culture flask (Greiner Bio-One, Frickenhausen, Germany). After 2  days of culture in an incubator at 5% CO2 and 37°C with standard medium, non-adherent cells were removed and the attached cells were washed twice with phosphate buffered saline (PBS). Further cultivation

was performed until the cells reached 70–80% confluency after 10–14 days. Cell detachment was achieved with 0.25% trypsin containing 1  mM EDTA (PAA) prior to collagen hydrogel fabrication.

MSC collagen hydrogel fabrication and culture Collagen type I (Col I) was derived from rat tails. The collagen solution was provided by Arthro Kinetics plc (Krems, Austria) at a concentration of 6 mg/ml. This stock solution was stored at -20°C. The MSCs were suspended in a gel neutralization solution (Arthro Kinetics) consisting of HEPES-buffered, double concentrated DMEM, and 20% FBS. The collagen stock solution was added at a ratio of 1:1. Subsequently, MSC collagen hydrogels with a volume of one ml were pipetted into 50 ml conical tubes. Gels with a cell density of 5 × 105 and 1 × 106 cells/ml and a collagen concentration of 3  mg Col I/ml were produced, respectively. For polymerization the gels were placed in an incubator for 30 min at 5% CO2 and 37°C. The cell laden hydrogels were cultured with different culture media in an incubator at 5% CO2 and 37°C as described in Table 1. The chondrogenic differentiation medium [28] consisted of high glucose DMEM (4.5  g/l, PAA) supplemented with dexamethasone (10 nM, Sigma), ascorbate 2-phosphate (50 μg/ml, Sigma), pyruvate (100 μg/ml, Sigma), proline (40 μg/ml, Merck, Darmstadt, Germany), antibiotics (50 IU penicillin/ml and 50  μg streptomycin/ml, PAA), glutamine (200 mM, PAA) and ITS-plus (final concentration: 6.25 μg/ml bovine insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.33 μg/ml linoleic acid and 1.25 mg/ml bovine serum albumin, Sigma). To initiate predifferentiation TGF-β1 was added (10 ng/ml, R+D, Wiesbaden, Germany). Media changes were performed every 3 days.

Table 1 Summary of the experimental groups included in the study.

Group 1 Group 2 Group 3 Group 4 Group 5



Culture media   day 1–10

Culture media day 11–21

         

SCM CDM-TGF-β1 CDM+TGF-β1 CDM+TGF-β1 CDM+TGF-β1

SCM CDM-TGF-β1 SCM CDM-TGF-β1 CDM+TGF-β1

         

SCM, stem cell medium; CDM, chondrogenic differentiation medium.

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F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels      377

Histochemical analysis Histochemical analyses were performed after 21 days for all groups. Briefly, the constructs were rinsed twice with PBS, fixed in PBS-buffered 4% paraformaldehyde for 2 h, dehydrated through an ascending series of ethanol, infiltrated with isoamyl acetate (Merck) and embedded in paraffin (Roth, Karlsruhe, Germany). Sections of thickness 4 μm were cut through the center of the constructs and stained with hematoxylin-eosin (H&E) and alcian blue.

RNA isolation and RT-PCR analysis RT-PCR analysis was carried out as described previously [17]. Each of the three samples was analyzed in triplicate per group and condition. Briefly, cells were centrifuged to pellets at day 0 and constructs were minced with scissors at day 21 after rinsing with PBS. RNA was extracted using the NucleoSpin® RNA II Kit (Macherey-Nagel, Düren, Germany). The RNA concentration was measured by spectrophotometry. Afterwards, the extracted RNA was converted to cDNA using dNTPs (10 mM, Bioline, Luckenwalde, Germany) and bioscript reverse transcriptase (200 U/μl, Bioline). Primers (MWG-Biotech, Ebersberg, Germany) were chosen to detect mRNA transcripts characteristic of hyaline cartilage. Elongation factor 1α served as a housekeeping gene (EF1α, Table 2). Aliquots containing 1 μl of the cDNA products were amplified using a Thermal Cycler (PTC-200, MJ Research, Waltham, MA, USA) in the presence of Taq polymerase (5000 U/ml, Bioline) at an initial denaturation at 94°C for 3 min followed by different numbers of cycles, each consisting of 45 s at 94°C, 1 min at a primer specific annealing temperature, 1 min extension at 72°C and a final incubation at 72°C for 5 min. The numbers of cycles and annealing temperatures were adjusted for each primer separately

to obtain the highest possible sensitivity and specificity of the reaction (Table 2). Some 15 μl DNA of each PCR reaction were electrophoretically separated using a 1.5% agarose gel containing ethidium bromide. DNA was visualized using imaging software (LTF, Wasserburg, Germany).

Results Culture and contraction of the collagen constructs After polymerization and addition of 3 ml culture medium, the conical tubes were gently agitated manually to detach the hydrogels. The gels had adopted the conical shape of the polypropylene tube. Over the culture period of 3 weeks a contraction was observed to result in more round gel spheroids, depending on the type of culture medium and cell number. While the negative controls without a differentiation factor showed only a contraction of 23% (SCM, 5 × 105 MSC/ml) to 47% (chondrogenic differentiation medium, 1 × 106 MSC/ml) of the original size, culture with chondrogenic differentiation medium and TGF-β1 (positive control) led to a severe contraction of up to 69% independent of the cell number. Chondrogenic predifferentiation with TGF-β1 over 10 days led to a contraction of 65% (5 × 105 MSC/ml) to 69% (1 × 106 MSC/ml). In contrast, further cultivation after 10  days using SCM resulted in slightly less contraction of 58% (5 × 105 MSC/ml) to 62% (1 × 106 MSC/ml).

Histochemical analysis Staining with H&E after 3 weeks of culture revealed homogeneously distributed cells within the hydrogel. A layer of more elongated cells was located at the hydrogel

Table 2 RT-PCR primer-sequence, product size, annealing temperature and amplification cycle number for each analyzed gene. Gene 

RT-PCR primer-sequence (5′–3′)



Product size (bp)



Annealing temperature (°C)

EF1α     AGN     Col II     Col X    

Sense: AGGTGATTATCCTGAACCATCC   Antisense: AAAGGTGGATAGTCTGAGAAGC   Sense: GCCTTGAGCAGTTCACCTTC   Antisense: CTCTTCTACGGGGACAGCAG   Sense: GAACATCACCTACCACTGCAAG   Antisense: GCAGAGTCCTAGAGTGACTGAG   Sense: CCCTTTTTGCTGCTAGTATCC   Antisense: CTGTTGTCCAGGTTTTCCTGGCAC 

235

               

54

392 374 468

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54 58 54

  Amplification cycle number                

26 40 40 32

378      F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels surface, especially in the TGF-β1 treated groups, depending on the degree of contraction. However, gels cultured with SCM were of lower cell numbers when compared to the other groups. Chondrocyte-like cells with of a round morphology embedded in surrounding lacunas could be detected in the predifferentiated (group 3 and 4) and positive control groups (group 5) independent of the cell

number. Interestingly, these cells were also seen in negative control constructs (group 1 and 2) although to a lower extend (Figure 1). Alcian blue staining performed after 3 weeks was negative in the control groups (groups 1 and 2) and showed a typical light homogeneous blue background staining, as known for collagen type I hydrogels. Constructs cultured

Figure 1 H&E staining of collagen hydrogels containing 5 × 105 (A, C, E, G, I) and 1 × 106 (B, D, F, H, J) MSC/ml after 3 weeks of culture using standard medium containing 10% FBS (A, B), chondrogenic differentiation medium without TGF-β1 (C, D), chondrogenic predifferentiation medium with 10% FBS (E, F), chondrogenic predifferentiation medium without 10% FBS (G, H) and chondrogenic differentiation medium with TGF-β1 (I, J). Bars 20 μm.

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F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels      379

with TGF-β1 showed a more intense staining around the chondrocyte-like cells, especially within the cell layer found at the surface, indicating that the cells were surrounded by a negatively charged proteoglycan-rich extracellular matrix (ECM). Highest stain intensity was observed in group 5 (Figure 2).

RT-PCR analysis RT-PCR analysis showed chondrogenic differentiation of MSCs in collagen type I hydrogels after 3 weeks of culture with TGF-β1 (group 5) as indicated by the expression of chondrogenic marker genes like collagen type II (Col II)

Figure 2 Alcian blue staining of collagen hydrogels containing 5 × 105 (A, C, E, G, I) and 1 × 106 (B, D, F, H, J) MSC/ml after 3 weeks of culture using standard medium containing 10% FBS (A, B), chondrogenic differentiation medium without TGF-β1 (C, D), chondrogenic predifferentiation medium with 10% FBS (E, F), chondrogenic predifferentiation medium without 10% FBS (G, H) and chondrogenic differentiation medium with TGF-β1 (I, J). Bars 50 μm.

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380      F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels and aggrecan. Compared to undifferentiated MSCs at day 0, chondrogenic predifferentiation with TGF-β1 over 10  days also caused up-regulation of Col II and aggrecan after 3 weeks (group 4). Interestingly, no expression of these markers was detected in hydrogels cultured with SCM (group 1 and 3) or chondrogenic differentiation medium without TGF-β1 (group 2). The hypertrophic marker collagen type X (Col X) was also expressed in all groups treated with TGF-β1. Concerning different cell numbers used for construct fabrication, the same gene expression pattern could be detected during the culture period (Figure 3).

Discussion MACI represents an established approach for the treatment of focal articular cartilage lesions. To circumvent the disadvantages associated with autologous chondrocyte isolation and expansion, orthopedic research has concentrated on MSCs as an alternative cell type.

Although it could be shown that chondrogenic differentiation of MSCs can be achieved, many factors that influence MSC proliferation and differentiation are still unknown. Nevertheless, a positive influence on the induction of a chondrogenic pathway was proven for growth factors of the TGF-β superfamily [5, 20, 21]. ­Consequently, stimulation of high density pellet cultures with TGF-β administered in a chemically well-defined differentiation medium resulted in chondrocyte-like cells surrounded by a proteoglycan-rich ECM expressing Col II [20, 29, 30]. Furthermore, it was demonstrated that MSCs are capable of proliferating and differentiating within three-dimensional matrices depositing sulfated glycosaminoglycans and expressing characteristic chondrogenic marker genes [28, 37]. A number of different materials have been investigated for cell-based cartilage repair. These include alginate, gelatine or hyaluronic-based scaffolds [29]. It is known that biomaterial composition and physicochemical and mechanical properties of these materials greatly influence nutrient supply and cellular behavior including

Figure 3 Expression of aggrecan (AGN), collagen type II (Col II) and X (Col X) at day 0 and day 21. Gels of two cell concentrations (5 × 105 and 1 × 106 MSCs/ml) were analyzed. Cell-hydrogel constructs were cultured in standard medium containing 10% FBS (1), chondrogenic d ­ ifferentiation medium without TGF-β1 (2), chondrogenic predifferentiation medium with 10% FBS (3), chondrogenic predifferentiation medium without 10% FBS (4) and chondrogenic differentiation medium with TGF-β1 (5). Shown is a representative analysis from a 63-year-old female.

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F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels      381

proliferation and differentiation. Despite continuing efforts, the perfect scaffold to deliver cells into the defect area has not yet been found [19]. For cell delivery to cartilage defects, collagen-based hydrogels in particular appear most suitable. Collagen-based biomaterials do not evoke immunogenic reactions, their biomechanical properties can easily be adjusted to specific needs, and they promote the attachment and proliferation of adhesive cell types. The CaReS®-system (Arthro Kinetics plc, Krems, Austria) represents a routinely applied system for MACI [2, 31, 32]. It uses three-dimensional collagen type I hydrogels for cell delivery. Previous studies could show that these hydrogels facilitate chondrogenic differentiation to a higher degree when compared to other materials [1]. In the present study, we aimed to achieve a chondrogenic predifferentiation within 10  days as this time frame resembles the chondrocyte expansion process in  vitro prior to transplantation when performing MACI with CaReS®. The objective was to initiate a chondrogenic pathway under controlled ex vivo conditions closely imitating physiologic conditions in the following by culturing cell-hydrogel constructs without a differentiation factor. This might further improve outcomes and reduce the degree of fibrocartilagenous repair tissue in favour of hyaline cartilage. As there is ample debate whether to transplant fully in vitro differentiated constructs or rather undifferentiated cells [18] this approach seems to be a good compromise. Recently, chondrogenic predifferentiation over 14 days of culture with TGF-β3 as a differentiation inducing factor was investigated by Marquass et  al. [22] using an ovine model. They could detect partially superior histological results of predifferentiated ovine MSC hydrogels compared with articular chondrocytes after 1 year of implantation. Furthermore, the repair tissue generated by predifferentiated constructs showed superior bonding to the surrounding cartilage tissue with no signs of degradation, cell hypertrophy or ectopic calcification. Indeed, all conclusions were drawn from histologic analyses. Nevertheless, these results may still indicate that gel contraction and Col X expression play a less important role in physiological conditions in vivo after transplantation. Compared to undifferentiated MSCs at day 0, we found an up-regulation of Col II and aggrecan after 3 weeks with TGF-β1 mediated chondrogenic predifferentiation over 10 days. Interestingly, this expression of chondrogenic markers was not observed when the predifferentiated MSCs were further cultured with SCM. These observations are in line with other studies that report the missing induction of chondrogenesis in rabbit bone marrow derived cells cultured in medium containing fetal bovine serum [20]. Conversely, histologically,

chondrocyte-like cells of round morphology were detected in the control group. These cells, however, did not express chondrogenic markers, which is consistent with previous outcomes published by Nöth et al. [28]. This phenomenon might be explained by the spontaneous chondrogenic differentiation of a subpopulation of MSCs and the induction of chondrogenic differentiation by the collagen hydrogel. Constructs cultured with SCM histologically seemed to be of lower cell numbers compared to the other groups also depending on the collagen contraction of the gels. This has been described in former investigations, which detected a slightly higher fraction of necrotic cells in the SCM control group [28]. In addition, potential MSC migration within the engineered constructs could be shown in recent studies [15, 16]. Nevertheless, further studies are necessary to analyze apoptosis and cell viability. In clinical practice, the contraction of the engineered constructs would represent one of the major problems related to transplant integration into the cartilage tissue. Compared to a previous study, we observed a similar degree of contraction of up to 69% in the TGF-β1 treated groups. This was independent of the cell number [28]. Consequently, hydrogel contraction seems to be closely related to growth factors used for differentiation as well as serum and gel collagen concentrations. Hydrogel contraction might be decreased by the use of short collagen fibers also resulting in higher gel permeability and cell viability. Furthermore, collagen cross-linking has previously been described as a possible way to reduce hydrogel contraction also improving their mechanical properties [24]. The limitations of this study include the small sample size and that all conclusions were drawn only from histochemical analyses and semi-quantitative RT-PCR. Future work will have to concentrate on the improvement of culture conditions which for example could include intermittent hydrostatic pressure or decreased oxygen supply as other groups could show the benefit of these modifications [4, 8, 10]. Furthermore, cell-free approaches might represent an option for cartilage repair. Recent studies investigating the repair of small cartilage defects in a Goettinger mini-pig using a cell-free collagen gel showed hyaline-like repair tissue of good quality and cellular in-growth without a known cell source. Outcomes were equivalent to a cell-based approach after 1 year [14, 33]. On the contrary, yet another study revealed no increase of repair tissue after microfracturing in chondral defects but when matrices seeded with autologous cells were applied in combination with microfracturing [11]. This illustrates that further investigations are necessary to address essential questions such as whether to use constructs with or without undifferentiated

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382      F. Fensky et al.: Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels respectively differentiated cells or whether to combine these approaches to solve issues related to cell source and donor site morbidity. In summary, the results of this study demonstrate that an ex vivo predifferentiation over a period of 10 days can induce chondrogenic differentiation of human MSCs embedded in collagen type I hydrogels. Further animal and clinical studies have to reveal whether this 10 day ex vivo predifferentiation can lead to a sufficient longterm stability and improvement of regenerated articular cartilage. Acknowledgments: The authors would like to acknowledge Arthro Kinetics plc for supplying the hydrogels.

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Chondrogenic predifferentiation of human mesenchymal stem cells in collagen type I hydrogels.

Hyaline cartilage displays a limited regenerative potential. Consequently, therapeutic approaches have been developed to treat focal cartilage lesions...
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