Annals of Biomedical Engineering, Vol. 42, No. 12, December 2014 ( 2014) pp. 2562–2576 DOI: 10.1007/s10439-014-1099-0

Initial Boost Release of Transforming Growth Factor-b3 and Chondrogenesis by Freeze-Dried Bioactive Polymer Scaffolds JAN PHILIPP KRU¨GER,1 ISABEL MACHENS,1 MATTHIAS LAHNER,2 MICHAELA ENDRES,1,3 and CHRISTIAN KAPS1,3 1 TransTissue Technologies GmbH, Charite´platz 1, 10117 Berlin, Germany; 2Department of Orthopaedic Sports Surgery, St. Josef-Hospital, Ruhr-University Bochum, Gudrunstr. 56, 44791 Bochum, Germany; and 3Tissue Engineering Laboratory, Department of Rheumatology, Charite´ - Universita¨tsmedizin Berlin, Charite´ Campus Mitte, Charite´platz 1, 10117 Berlin, Germany

(Received 12 May 2014; accepted 23 August 2014; published online 29 August 2014) Associate Editor Kent Leach oversaw the review of this article.

INTRODUCTION

Abstract—In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to analyze the chondrogenic potential of freeze-dried resorbable polymerbased polyglycolic acid (PGA) scaffolds bio-activated with transforming growth factor-b3 (TGFB3) on human subchondral mesenchymal progenitor cells known from microfracture. Progenitor cells derived from femur heads were cultured in the presence of freeze-dried TGFB3 in highdensity pellet culture and in freeze-dried TGFB3-PGA scaffolds for chondrogenic differentiation. Progenitor cell cultures in PGA scaffolds as well as pellet cultures with and without continuous application of TGFB3 served as controls. Release studies showed that freeze-dried TGFB3-PGA scaffolds facilitate a rapid, initial boost-like release of 71.5% of TGFB3 in the first 10 h. Gene expression analysis and histology showed induction of typical chondrogenic markers like type II collagen and formation of cartilaginous tissue in TGFB3-PGA scaffolds seeded with subchondral progenitor cells and in pellet cultures stimulated with freeze-dried TGFB3. Chondrogenic differentiation in freeze-dried TGFB3-PGA scaffolds was comparable to cultures receiving TGFB3 continuously, while non-stimulated controls did not show chondrogenesis during prolonged culture for 14 days. These results suggest that bio-activated, freeze-dried TGFB3PGA scaffolds have chondrogenic potential and are a promising tool for stem cell-mediated cartilage regeneration.

Traumatic or degenerated focal cartilage defects occur frequently and may progress to severe osteoarthritis11 and eventually lead to total replacement of the respective joint. Therefore, aiming at regenerating the cartilage and restoring its surface, several surgical cartilage repair procedures comprising e.g. bone marrow stimulating techniques, osteochondral autograft transfer and autologous chondrocyte implantation (ACI) have been developed.5,7,31,64 In particular bone marrow stimulating techniques like drilling or microfracturing are easy to apply, cost effective and therefore frequently used first-line treatment options for cartilage defects. In microfracture, the defective cartilage is removed and multiple ‘fractures’ are introduced into the subchondral bone using a chondropick awl. Bleeding into the defect occurs accompanied by an influx of mesenchymal stem or progenitor cells that form cartilaginous repair tissue filling the defect. Stem cell in-growth as well as formation of cartilage repair tissue may be supported or stimulated by growth and differentiation factors from the subchondral bone and/or the synovial fluid.17,58,63 Clinical studies showed that the microfracture technique leads to a good clinical outcome in the short and mid-term with up to 5 years follow-up, but the newly formed repair tissue is often of a fibrocartilaginous appearance with limited short-term durability.28,40,41 However, in the long-term the outcome is worse with osteoarthritis and treatment failures at 5–10 years follow-up, regardless of defect sizes.27 It is assumed that formation and/or regeneration of hyaline cartilage may overcome those long-term

Keywords—Cartilage tissue engineering, Cartilage regeneration, Resorbable polymer scaffold, Polyglycolic acid, Chondrocyte differentiation, Freeze-drying, Growth factor release.

Address correspondence to Christian Kaps, TransTissue Technologies GmbH, Charite´platz 1, 10117 Berlin, Germany. Electronic mail: [email protected]

2562 0090-6964/14/1200-2562/0

 2014 Biomedical Engineering Society

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

obstacles and, therefore, improvement of the microfracture technique is demanded that may enhance the content or composition of key cartilage matrix components.23 Thus, improvement may lead to better and more durable cartilaginous repair tissue. To achieve such an improvement of the microfracture technique, recent developments favor covering the microfractured defects with e.g. porcine collagen scaffolds with fibrin,3,26 chitosan-based gels62 or bio-activated, polymer-based polyglycolic acid-hyaluronan (PGA-HA) scaffolds.50,69 Covering of the defect may thereby lead to an enrichment of progenitor cells in the defect and improved repair tissue formation. In the ovine joint defect model, the use of PGA-HA scaffolds bio-activated with autologous serum for covering microfractured defects has been shown to lead to better cartilaginous repair tissue of a hyaline-like cartilage appearance with collagen type II deposition than found in microfracture alone.20 The resorbable polyglycolic acid-based scaffold is a non-woven, textile feltlike structure made of pure polyglycolic acid polymer fibers of about 17 lm in thickness. The stiffness of the scaffold is equivalent to native human cartilage and its tensile strength is reduced by approx. 50% during the first 10 days of degradation in liquids.18 The hyaluronan component of the PGA-HA scaffold with a molecular weight of approx. 1200 kDa has been shown to induce chondrogenic differentiation of mesenchymal stem cells in an equine in vitro model and is incorporated into the PGA by freeze-drying.21,29 From the clinical point of view, safety and efficacy of using the PGA-HA scaffold for cartilage repair has been shown in a case series including 52 patients. Implantation of the scaffolds bio-activated with autologous plateletrich plasma significantly improved the patients’ situation as shown by the Knee injury and Osteoarthritis Outcome Score (KOOS) at one and 2-year follow-up and formed hyaline-like cartilage tissue as assessed by second-look biopsies.59,60 Bio-activation of scaffolds for cartilage repair with patients’ own blood-derived components like serum or platelet-rich plasma (PRP) during surgery is not standardized, is e.g. accompanied by achieving non-defined growth factor contents in the scaffold and may therefore lead to a hardly predictable clinical outcome. However, blood-derived components like PRP are well known sources of autologous growth factors that eventually support the cartilage repair sequence by e.g. having anti-inflammatory and/or chondrogenic effects.54 Recently, it has been shown that members of the transforming growth factor family of growth and differentiation factors that are known to have chondrogenic potential, in particular transforming growth factor-b3 (TGFB3), are robustly present in human PRP and are likely involved in chondrogenic

2563

differentiation of stem and progenitor cells during cartilage repair.42 In a variety of studies it has been shown that continuous and repeated administration of 10 ng/mL TGFB3 induces chondrogenic differentiation of mesenchymal stem and progenitor cells derived from different species in vitro.22,46,57 For in vivo approaches in cartilage repair a broad range of doses with 50–250 ng or 100 ng/mL up to 5 lg/mL TGFB3 has been reported.33,36,67 However, the application of high doses (>900 ng/mL TGFB3) has been associated with side effects like e.g. synovitis and cartilage erosion.67 The aim of our present study was to evaluate the PGA scaffold bio-activated with the chondrogenic inducer TGFB3 followed by freeze-drying, with regard to its potential to stimulate chondrogenic differentiation of human subchondral mesenchymal progenitor cells known from microfracturing and to controlled release of the growth factor. Taking dose ranges for chondrogenesis and cartilage repair from literature into account, cultures repeatedly treated with 10 ng/mL TGFB3 served as positive controls, while single doses of 300 and 600 ng TGFB3 were used in bio-activation and subsequent freeze-drying of PGA scaffolds, corresponding to 200 ng/mL TGFB3 for the in vitro chondrogenic differentiation assays.

MATERIALS AND METHODS Isolation and Culture of Human Subchondral Mesenchymal Progenitor Cells Human subchondral mesenchymal progenitor cells were obtained from femur heads of 8 donors post mortem (1 female, 7 males, mean age 65 years, range 58–79 years). Macroscopically, there were no signs of cartilage lesions or osteoarthritic degeneration. Progenitor cells were harvested as described previously.49 In brief, spongious bone of the femur heads was cut into small fragments, digested for 4 h at 37 C using 256 U/mL collagenase XI (Sigma-Aldrich, USA), placed in PrimariaTM cell culture flasks (Becton and Dickinson, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Biochrom, Germany) containing 10% human serum (German Red Cross, Berlin, Germany), 100 U/mL penicillin, 100 lg/mL streptomycin, 100 lg/mL gentamycin, 0.1 lg/mL amphotericin B (all Biochrom) and 2 ng/mL human fibroblast growth factor-2 (PeproTech, Germany). At 80–90% confluency, cells were sub-cultivated using trypsin in phosphate-buffered saline (0.05% v/v, Biochrom) and re-plated at a density of 8000 cells/cm2. Medium exchange was performed every 2 or 3 days. The ethics committee of the Charite´-Universita¨tsmedizin Berlin approved the study.

2564

KRU¨GER et al.

Chondrogenic Stimulation of Human Subchondral Mesenchymal Progenitor Cells with Freeze-Dried TGFB3 in High-Density Pellet Culture Chondrogenic stimulation of human subchondral mesenchymal progenitor cells (passage 3) was performed under serum free conditions in high-density pellet cultures (pool of cells of n = 3 donors, 3 males, mean age 71 years, range 67–79 years, 250,000 cells/ pellet), as described previously.35 Human recombinant transforming growth factor-b3 (TGFB3, R&D Systems, USA) was rehydrated according to the manufacturer’s recommendations, frozen at 220 C for 30 min and immediately freeze-dried for 16 h using a freeze-dryer (Heto Powerdry LL1500, Thermo Electron, Germany) with a condenser temperature of 2110 C. Cell pellets were stimulated by adding 10 ng/ mL TGFB3 (n = 43 pellets) and 100 ng/mL (n = 43 pellets) as well as 200 ng/mL (n = 43 pellets) freezedried TGFB3 in complete DMEM containing 1% ITS + 1 (Insulin-Transferrin-Selenium; BD Biosciences, Germany), 1 mM sodium pyruvate, 0.35 mM L-proline, 0.17 mM L-ascorbic acid-2-phosphate and 0.1 lM dexamethasone (all Sigma-Aldrich, USA). Cell pellets (n = 43) cultured in complete DMEM without the addition of TGFB3 served as controls. Cell pellets were kept in the respective medium for four days to allow for pellet formation, followed by complete removal of the supernatants and further culture in complete DMEM with 10 ng/mL TGFB3 (TGFB3 cont - group) or complete DMEM without TGFB3 (controls, TGFB3 boost [100 ng/mL] group and TGFB3 boost [200 ng/mL] group). Gene expression analysis was performed at day 2 (n = 20 pellets per group) and day 4 (n = 20 pellets per group), while histological staining was performed at day 7 (n = 3 pellets per group).

Chondrogenic Differentiation of Human Subchondral Mesenchymal Progenitor Cells in Freeze-Dried TGFB3Polyglycolic Acid Scaffolds For preparation of freeze-dried TGFB3-polyglycolic acid (TGFB3-PGA) scaffolds, polyglycolic acid scaffolds (n = 15, 10 mm 9 5 mm 9 1.1 mm, Soft PGA Felt, Alpha Research Deutschland GmbH, Germany) were immersed with 600 ng TGFB3 solved in 55 lL BSA-PBS (1% bovine serum albumin in PBS) per scaffold and freeze-dried for 16 h using a freezedryer (Heto Powerdry LL1500, Thermo Electron, Germany). Human subchondral mesenchymal progenitor cells (n = 5 donors, 1 female, 4 males, mean age 62 years, range 58–67 years, passage 3) were re-suspended in fibrinogen (33% [volume/volume] fibrinogen [Tissucol,

Baxter, Germany] in complete DMEM) at a cell density of 2.4 9 107 cells/mL. Freeze-dried TGFB3-PGA scaffolds and pure PGA scaffolds were immersed with the cell-fibrinogen suspension and the fibrinogen was polymerized by addition of thrombin (1:10 in PBS; Tissucol; Baxter, Germany). Subsequently, TGFB3PGA-fibrin progenitor grafts (designated TGFB3 boost, n = 3 per donor, n = 15 in total) were cultured in 3 mL of complete DMEM, being equivalent to a TGFB3 concentration of 200 ng/mL. PGA-fibrin progenitor grafts (designated TGFB3 cont, n = 3 per donor, n = 15 in total) were cultured in 3 mL complete DMEM supplemented with 10 ng/mL TGFB3, while PGA-fibrin progenitor grafts (designated control, n = 3 per donor, n = 15 in total) cultured in 3 mL complete DMEM without TGFB3 served as controls. Grafts were kept in the respective medium for 4 days without exchange of medium. Medium exchange was performed at day four and every other day thereafter. TGFB3 Release Kinetics of Freeze-Dried TGFB3Polyglycolic Acid Scaffolds TGFB3 release kinetics were obtained in the presence of fibrin as well as with and without medium exchange, in triplicates each. TGFB3-PGA scaffolds (5 mm 9 5 mm 9 1.1 mm, loaded with 300 ng TGFB3) were prepared (n = 66, see above), loaded with 30 lL fibrinogen (33% [volume/volume] fibrinogen [Tissucol, Baxter, Germany] in BSA-PBS (1% bovine serum albumin in PBS)), polymerized by addition of thrombin (1:10 in PBS; Tissucol; Baxter, Germany). For measurement of TGFB3 release data without medium exchange, scaffolds (n = 45) were kept in 1.5 mL BSA-PBS up to 14 days, being equivalent to a TGFB3 concentration of 200 ng/mL. To mimic cell culture conditions used for chondrogenic differentiation of mesenchymal progenitor cells in TGFB3-PGA scaffolds, scaffolds (n = 21) were also kept in 1.5 mL BSA-PBS for 4 days without medium exchange, were subjected to complete medium exchange at day 4 and every other day thereafter. The respective supernatants were stored at 280 C. TGFB3 content in supernatants was measured in triplicates with TGFB3 sandwich enzyme-linked immunosorbent assay (ELISA) kit (DuoSet ELISA Development Kit, Cat.No. DY243, R&D Systems, USA) according to the manufacturer’s instructions. In brief, the ELISA assay was performed in a 96-well plate pre-coated with a monoclonal mouse anti-human TGFB3 capture antibody. Samples (100 lL) were applied in triplicates and incubated for 2 h at room temperature. After washing with 0.05%Tween in phosphate buffered saline (PBS), biotinylated polyclonal goat anti-human TGFB3 detection antibody

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

was added and incubated for 2 h at room temperature, followed by washing steps and incubation with streptavidin coupled with horseradish peroxidase (HRP). Conversion of the substrate (H2O2—tetramethylbenzidine) was measured using a microplate reader at 450 nm and wavelength correction at 540 nm. TGFB3 levels in the respective supernatants were calculated from a standard curve of 0–2000 pg/mL TGFB3. Activation of latent TGFB3 was not performed. According to the manufacturer, the ELISA detects mature TGFB3. There is no information whether the ELISA system distinguishes between biologically active and non-active TGFB3. The ELISA system is calibrated against highly purified recombinant human TGFB3 expressed by Sf21 cell lines and detects TGFB3 in the range of 31.2–2000 pg/mL. For elimination of outliers due to technical issues, confidence interval (CI) was calculated and data beyond were analysed using the Grubbs outlier test. The actual release of TGFB3 per volume (ng/mL) between consecutive points in time (e.g. between 6 h and 10 h after start of the release experiment) was calculated from the data/concentrations of TGFB3 found in the supernatant by subtracting the concentration found in the supernatant at the preceding from the latter time point (e.g. release between 6 h and 10 h = concentration of TGFB3 in the supernatant at 10 h minus concentration of TGFB3 in the supernatant at 6 h). Reproducibility of Freeze-Dried TGFB3-Polyglycolic Acid Scaffolds To determine the recovery rate of TGFB3 after loading in PGA scaffolds and subsequent freeze-drying, PGA scaffolds (n = 12; 5 mm 9 5 mm 9 1.1 mm) were loaded with 300 ng TGFB3 in 25 lL BSA-PBS (1% bovine serum albumin in PBS) and freeze-dried (n = 2 independent freeze-drying cycles) as described (see ‘‘Chondrogenic Stimulation of Human Subchondral Mesenchymal Progenitor Cells with Freeze-Dried TGFB3 in High-Density Pellet Culture’’ section). Freezedried TGFB3-PGA scaffolds were rehydrated by adding 1 mL BSA-PBS and incubation at room temperature for 10 min. After harvesting the supernatant, the remaining rehydrated TGFB3-PGA scaffold was centrifuged at 720 g for 10 min. The eluate of the centrifuged TGFB3PGA scaffold and the respective supernatant were pooled and the TGFB3 content was measured by ELISA (see ‘‘TGFB3 Release Kinetics of Freeze-Dried TGFB3Polyglycolic acid Scaffolds’’ section). Non-freeze dried TGFB3 (300 ng) in 1 mL BSA-PBS (n = 2) served as control, representing 100% TGFB3. The mean recovery rate was determined by calculating the amount of TGFB3 in relation to controls, including standard deviation and variation coefficient.

2565

Polymerase Chain Reaction (PCR) Total RNA from high-density pellet cultures (n = 20 per point in time and experimental condition) and from TGFB3-PGA, PGA-fibrin and control progenitor grafts (n = 1 per donor, n = 5 in total per point in time and experimental condition) was isolated as described previously9 and 1 lg RNA was reversely transcribed with iScript cDNA Synthesis Kit according to the manufacturer’s recommendations (BioRad, Germany). The relative expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the samples. Realtime RT-PCR was performed with 1 lL of each cDNA sample in triplicates using i-Cycler PCR system (Bio BioRad, Germany) with SYBR green PCR Core Kit (Applied Biosystems, USA). Relative quantification of marker genes (Table 1) was performed and is given as percentage of the GAPDH product.

Histology and Immunohistochemistry For histological staining, high-density pellets were embedded in OCT compound, frozen and cryo-slides (6 lm, n = 3 per point in time and experimental conditions) were prepared. Proteoglycans were visualized by staining with Alcian Blue 8GS (Roth, Germany) at pH 2.5. Nuclei were counterstained using nuclear fast red (Merck, Germany). Progenitor cells in PGA and TGFB3-PGA scaffolds were embedded in OCT compound, frozen and cryo-slides (8 lm) were prepared. Proteoglycans were stained using Alcian Blue 8GS (Roth) at pH 2.5 (n = 5 donors, n = 3 slides per donor and experimental condition) and Safranin O (n = 5 donors, n = 3 slides per donor and experimental condition, Sigma-Aldrich, USA). Von Kossa staining and counterstaining with nuclear fast red were used to detect mineralization of the extracellular matrix (n = 5 donors, n = 3 slides per donor and experimental condition). For immunohistochemistry, cryo-slides (n = 5 donors, n = 3 slides per donor and experimental condition) were incubated for 40 min with primary antibodies (mouse anti-human type II collagen, Acris, Germany). Mouse IgG served as control (DAKO, Germany). Detection was performed according to the manufacturer’s instructions using the EnVision ++ System HRP (AEC) Kit (DAKO, Germany), followed by counterstaining with hematoxylin. Histomorphometric analysis was carried out as published previously.43 In brief, quantification of extracellular matrix formation as assessed by Alcian blue (n = 5 donors, n = 3 slides per donor and experimental condition), Safranin O (n = 5 donors, n = 3 slides per donor and experimental condition) and type II collagen (n = 5 donors, n = 3 slides per

2566

KRU¨GER et al. TABLE 1. Oligonucleotide sequences. Oligonucleotides (5¢ fi 3¢)(up/down)

Base pairs

NM_000088 NM_001844 NM_000493 NM_000095

CGATGGCTGCACGAGTCACAC/CAGGTTGGGATGGAGGGAGTTTAC CCGGGCAGAGGGCAATAGCAGGTT/CAATGATGGGGAGGCGTGAG GAACTCCCAGCACGCAGAATCC/GTGTTGGGTAGTGGGCCTTTTATG CCGGAGGGTGACGCGCAGATTGA/TGCCCTCGAAGTCCACGCCATTGAA

180 128 145 133

NM_001135 NM_002046

GGCTGCTGTCCCCGTAGAAGA/GGGAGGCCAAGTAGGAAGGAT GGCGATGCTGGCGCTGAGTAC/TGGTCCACACCCATGACGA

163 149

Gene name

Accession No.

Collagen type I Collagen type II Collagen type X Cartilage oligomeric matrix protein Aggrecan Glyceraldehyde-3-phospate dehydrogenase

donor and experimental condition) staining was performed using the Photoshop software. The region of interest (ROI) of a given slide to be analyzed (Fig. 1a) was defined by marking the background (Fig. 1b, black area). The amount of pixels was measured within the ROI (Fig. 1c, red area) using the histogram function. A distinct standard was defined, which represents the particular color for the specific staining. The tools ‘‘magic wand’’ and ‘‘select similar’’ were used to select areas of that particular color (Fig. 1d showing selected areas per staining in red avoiding counting background (asterisks), scaffold fibres (black arrowhead) and cells (black double arrowhead)). Subsequently, the percentage of the stained pixels (particular color per staining) in relation to the total number of pixels of ROI was calculated. Statistical Analysis For analysis of histomorphometric and gene expression values the SigmaStat 3.5 software (Systat Software GmbH, Germany) was used. The Kolmogorov–Smirnov method was applied for testing normal distribution of the data. For normal distributed data, the t test was applied, while the Mann–Whitney rank sum test was used for data that failed normality testing. Differences were considered significant at p < 0.05.

RESULTS Freeze-Dried TGFB3 is Biologically Active and Induces Chondrogenic Differentiation of Human Mesenchymal Progenitor Cells in High-Density Pellet Culture The recovery rate of TGFB3 after loading in PGA scaffolds, subsequent freeze-drying and rehydration was 99.9% with a standard deviation of 5.4% and a variation coefficient of 0.05. The biological activity of freeze-dried TGFB3 was assessed by evaluating its potential to stimulate chondrogenic differentiation of human mesenchymal subchondral progenitor cells in high-density pellet culture

(Fig. 2). Controls, mesenchymal progenitor cells that were cultured in the absence of TGFB3 for 7 days, formed a dense pellet with marginal formation of a proteoglycan-rich extracellular matrix (Fig. 2a). In the presence of 10 ng/mL TGFB3 and renewal of the medium after 4 days of culture, mesenchymal progenitor cells showed compact pellets with a proteoglycan-rich extracellular matrix (Fig. 2b). The same applies to pellets that were initially stimulated once with 100 ng/mL (Fig. 2c) or 200 ng/mL (Fig. 2d) freeze-dried TGFB3. Gene expression analysis performed in the early phase of pellet formation at day 2 and day 4 confirmed chondrogenic differentiation of mesenchymal progenitors upon treatment with 10 ng/ mL TGFB3 and freeze-dried TGFB3 (Fig. 2e). At day 4 and compared with non-stimulated controls, mesenchymal progenitor cells stimulated with 10 ng/mL TGFB3 or once with 100 ng/mL and 200 ng/mL freeze-dried TGFB3 showed elevated levels of the chondrogenic marker genes aggrecan (in mean 1.1% of the expression level of GAPDH in controls to in mean 2.0–2.2% upon TGFB3 treatment), collagen type II (0.4% in controls to 4.4–10.2% upon TGFB3 treatment) and cartilage oligomeric matrix protein (COMP; 0.5% in controls to 4.6–9.6% upon TGFB3 treatment). Since gene expression values of chondrogenic marker genes were elevated in mesenchymal progenitor cells stimulated once with 200 ng/mL freeze-dried TGFB3 compared to values found for progenitors treated with 100 ng/mL freeze-dried TGFB3, further differentiation and release studies were performed with 200 ng/mL freeze-dried TGFB3. Formation of Cartilaginous Extracellular Matrix by Human Subchondral Mesenchymal Progenitor Cells in Freeze-Dried TGFB3-Polyglycolic Acid (PGA) Scaffolds Cartilaginous extracellular matrix formation was assessed by histological staining of proteoglycans with Alcian blue and Safranin O as well as by immunostaining of cartilage-specific collagen type II (Fig. 3).

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

2567

FIGURE 1. Histomorphometric image analysis of histological and immune-histochemical staining. The region of interest (ROI) of a given slide to be analyzed (a) was defined by marking and excluding the background (b, black area). The amount of pixels was measured within the ROI (c, red area), followed by selecting and measuring the number of pixels with the particular color for the specific staining (d, red area), while background (d, asterisks), scaffold fibres (d, black arrowhead) and cells (d, black double arrowhead) are excluded.

Controls of human mesenchymal progenitor cells cultured in PGA scaffolds in the absence of TGFB3 for 14 days did not form a cartilaginous extracellular matrix. The tissue was void of proteoglycans (Fig. 3a/ b) and collagen type II (Fig. 3c), while PGA scaffold fibers (Fig. 3a, black arrowhead), mesenchymal progenitor cells (Fig. 3a, white arrowhead) and a fibrous extracellular matrix (Fig. 3a, asterisk) were evident. In contrast, continuous and repeated addition of 10 ng/ mL TGFB3 (TGFB3 cont) to mesenchymal progenitor cells embedded in PGA scaffolds resulted in the formation of a cartilaginous extracellular matrix rich in proteoglycans (Fig. 3d/e) and collagen type II (Fig. 3f). Human subchondral mesenchymal progenitor cells cultured in freeze-dried TGFB3-PGA scaffolds without further addition of TGFB3 for 14 days (TGFB3 boost) formed cartilaginous extracellular matrix with proteoglycans (Fig. 3g/h) and collagen type II (Fig. 3i).

Quantification of cartilaginous matrix formation by histomorphometric analysis (Fig. 4) showed significantly higher amounts of proteoglycans with mean 84.2% of the scaffold area positively stained with Alcian blue in the ‘TGFB3 cont’ group and mean 78.3% in the ‘TGFB3 boost’ group, while in controls mean 4.0% of the scaffold area was stained (Fig. 4a). Compared to controls (mean 1.0%), the red proteoglycan staining by Safranin O was significantly increased in the ‘TGFB3 cont’ (mean 49.9%) and ‘TGFB3 boost’ (mean 38.2%) group, while the green staining indicative for collagenous, fibrous tissue was significantly reduced (Fig. 4b). Collagen type II (Fig. 4c) was significantly elevated in the ‘TGFB3 cont’ group (mean 81.5% positively stained area) and in the ‘TGFB3 boost’ group (mean 73.3%), compared to controls (mean 1.0%). There was no significant difference in proteoglycan and collagen type II staining between the ‘TGFB3 cont’ group receiving continuous

2568

KRU¨GER et al.

FIGURE 2. Chondrogenic differentiation of human subchondral progenitor cells in high-density pellet culture upon stimulation with freeze-dried TGFB3. Alcian blue proteoglycan staining of non-stimulated controls (a), high-density progenitor cell pellets continuously stimulated with 10 ng/mL TGFB3 (b) and after stimulation once with 100 ng/mL (c) and 200 ng/mL (d) freeze-dried TGFB3, at day 7. Chondrogenic differentiation of progenitor cell pellets was analyzed at day 2 and day 4 by gene expression analysis of typical chondrogenic marker genes aggrecan, collagen type I and cartilage oligomeric matrix protein (COMP). The expression level of marker genes was calculated as percentage of the expression level of the housekeeping gene GAPDH. The bars show the mean (n 5 3) and the SD.

and repeated addition of TGFB3 and the ‘TGFB3 boost’ group with freeze-dried TGFB3-PGA scaffolds and no further addition of TGFB3. Induction of Chondrogenic Marker Genes by Human Subchondral Mesenchymal Progenitor Cells in FreezeDried TGFB3-Polyglycolic Acid (PGA) Scaffolds Gene expression profiles of typical chondrogenic marker genes were determined throughout the culture period at day 4, day 7 and day 14 (Fig. 5). At day 14 and compared with controls, continuous and repeated addition of TGFB3 (TGFB3 cont) to mesenchymal progenitor cells in PGA scaffolds significantly increased expression levels of the marker genes aggrecan (from mean 1% related to the level found for the housekeeping gene GAPDH in controls to mean 4%) and collagen type II (mean 43% in controls to mean 923%). The expression of COMP remained stable (mean 72% in controls, mean 69% in ‘TGFB3 cont’ group) and expression of collagen type I being a marker for fibrous tissue was significantly decreased (mean 8648% in controls to mean 1175%). Culture of mesenchymal progenitor cells in freeze-dried TGFB3PGA scaffolds without further addition of TGFB3 (TGFB3 boost) for 14 days resulted in elevated levels of the marker genes aggrecan (mean 2%) and COMP (mean 90%) as well as significantly increased

expression levels of collagen type II (mean 43% in controls to mean 1894%). The expression of collagen type I was decreased in progenitor cells cultured in TGFB3-PGA scaffolds (mean 4287%). Analysis of Extracellular Matrix Mineralization and Chondrocyte Hypertrophy in Human Subchondral Mesenchymal Progenitor Cells Cultured in Freeze-Dried TGFB3-Polyglycolic Acid (PGA) Scaffolds Since ossification as well as chondrocyte hypertrophy is often associated with stem and progenitor cell chondrogenesis, mineralization of the extracellular matrix and the gene expression profile of the chondrocyte hypertrophy marker collagen type X were evaluated (Fig. 6). Von Kossa staining was negative and there were no signs for mineralization of the extracellular matrix formed by non-stimulated progenitors cultured in PGA scaffolds for 14 days (Fig. 6a, control), by progenitors in PGA scaffolds subjected to continuous and repeated stimulation with TGFB3 (Fig. 6b, TGFB3 cont) and by progenitors cultured in freeze-dried TGFB3-PGA scaffolds without further addition of TGFB3 (Fig. 6c, TGFB3 boost). At day 4 to day 14, the gene expression levels of collagen type X (Fig. 6d) were significantly increased in the ‘TGFB3 cont’ group (from mean 7% related to the level found for the housekeeping gene GAPDH in

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

2569

FIGURE 3. Histological staining of cartilaginous extracellular matrix formed by human subchondral progenitor cells cultured in freeze-dried TGFB3-PGA scaffolds. Formation of extracellular matrix by non-stimulated controls (a–c) with fibrous matrix (a, asterisk), PGA scaffold fibers (a, black arrowhead) and progenitor cells (a, white arrow head) as well as cartilaginous matrix formation by progenitor cells embedded in PGA scaffolds with continuous stimulation with 10 ng/mL non-freeze-dried TGFB3 (d–f) and by progenitors cultured in freeze-dried TGFB3-PGA scaffolds (g–i) as assessed by Alcian blue and Safranin O proteoglycan staining and immunostaining of cartilage-specific collagen type II.

controls to mean 322%, at day 14) and in the ‘TGFB3 boost’ group (mean 198%). During the course of culture, there was no significant difference between the expression levels of collagen type X in progenitors cultured in PGA scaffolds with continuous and repeated stimulation with TGFB3 and the progenitors cultured in freeze-dried TGFB3-PGA scaffolds. Release of TGFB3 from Freeze-Dried TGFB3Polyglycolic Acid (PGA) Scaffolds To evaluate release kinetics and stability of TGFB3 over time, the amount of released TGFB3 from freezedried TGFB3-PGA scaffolds was measured during a period of 14 days (Fig. 7a). TGFB3 is released from the TGFB3-PGA scaffold boost-like within the first 10 h of incubation. After 10 h, the concentration of TGFB3 in the supernatant reached the high level plateau with 172.5 ng/mL, which corresponds to a

boost-like release of 143 ng/mL TGFB3 from freezedried TGFB3-PGA scaffolds after 6–10 h (71.5% of TGFB3 incorporated in the scaffold). The concentration of TGFB3 in the supernatant remained stably on a high level plateau after 10 h to 14 days, ranging from 167.5 to 198.2 ng/mL TGFB3, while further release of TGFB3 was negligible low. TGFB3 remains stable and present in the supernatant during the course of 14 days at 37 C, but there is no information about its activity. To mimic cell culture conditions used in prolonged chondrogenic differentiation culture with regular medium exchange for cell nutrition, the supernatant was completely exchanged at day 4 and every other day thereafter, while the amount of TGFB3 in the supernatant released from freeze-dried TGFB3-PGA scaffolds was measured prior to removal (Fig. 7b). TGFB3 released from freeze-dried TGFB3-PGA scaffolds showed the highest level after 10 h to 4 days with 167.9 to 172.5 ng/mL TGFB3 in the supernatant. After first

2570

KRU¨GER et al.

DISCUSSION

FIGURE 4. Histomorphometric image analysis of cartilaginous extracellular matrix formed by human subchondral progenitor cells cultured in freeze-dried TGFB3-PGA scaffolds. Histomorphometric image analysis indicated that continuous stimulation of progenitor cells with 10 ng/mL nonfreeze-dried TGFB3 (TGFB3 cont) as well as culture of cells in freeze-dried TGFB3-PGA scaffolds (TGFB3 boost) resulted in cartilaginous extracellular matrix formation with significantly (*p < 0.05) higher percentages of stained areas for Alcian blue (a), Safranin O (b) and collagen type II (c) staining, compared to controls. The bars show the mean (n 5 15) and the SD.

complete removal and exchange of the supernatant the TGFB3 level dropped to 75.3 ng/mL at day 6 and to 0.2 ng/mL at day 14. Again, this corresponds to the first boost-like release after 6–10 h of 143 ng/mL TGFB3 and to a second boost-like release after day 4 to day 6 of 75.3 ng/mL TGFB3, while further release of TGFB3 from freeze-dried TGFB3-PGA scaffolds was low ranging from 8.1 to 0.2 ng/mL TGFB3.

In the present study, we have demonstrated that freeze-dried TGFB3 is bioactive and induces chondrogenic differentiation of human subchondral mesenchymal progenitor cells cultured in high-density pellets and resorbable polyglycolic acid (PGA) scaffolds. Freeze-dried TGFB3-PGA scaffolds show a rapid release of TGFB3 in terms of an initial boost, which is sufficient to induce the chondrogenic differentiation sequence of subchondral progenitor cells in vitro accompanied by induction of typical chondrogenic marker genes and formation of cartilaginous extracellular matrix rich in proteoglycans and cartilage-specific collagen type II. Cartilaginous matrix formation by subchondral progenitor cells in freezedried TGFB3-PGA scaffolds in vitro was equivalent to matrix formation by subchondral progenitor cells in conventional PGA scaffolds induced by continuous and repeated addition of TGFB3. Human mesenchymal stem cells derived from bone marrow (MSC) as well as human subchondral progenitor cells have a multi-lineage differentiation capacity and undergo chondrogenic differentiation upon treatment with TGFB.49,52 In particular, all TGFB isoforms have been shown to stimulate chondrogenesis in high-density pellet cultures.4,35 However, although there are no differences reported regarding the collagen deposition during chondrogenic stimulation with different TGFB isoforms in human MSC,8 the temporal availability of particular isoforms during the chondrogenic sequence of progenitor cells in vitro may be of importance. Recently, a time-dependent effect of TGFB1 mediated chondrogenic differentiation on MSC has been shown in poly-e-caprolactone (PCL) scaffolds. The collagen type II expression seems to be related to a continuous or late supply of TGFB1,45 while culture of MSC in PCL or PCL-hyaluronan scaffolds rapidly releasing TGFB1 in the early phase of chondrogenesis leads to lower levels of collagen type II.56 Continuous stimulation of human bone marrow-derived MSC with TGFB3 in vitro has been shown to promote chondrogenic differentiation in different scaffolds like e.g. chitosan-based chitosanpoly-butylene terephtalate adipate scaffolds,2 RGDconjugated poly(ethylene glycol) microspheres53 or polyglycolic acid-hyaluronan scaffolds.51 As shown in our current study, continuous as well as initial boost application of TGFB3 resulted in robust chondrogenic differentiation of human subchondral mesenchymal progenitor cells in PGA scaffolds without differences in collagen type II expression or deposition. These findings may advance TGFB3 for its use in bio-activation of scaffolds for cartilage repair. However, the induction of collagen type X by TGFB3 in our in vitro

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

2571

FIGURE 5. Semi-quantitative real-time gene expression analysis of chondrogenic marker genes. Chondrogenic differentiation of human subchondral mesenchymal progenitor cells was analyzed by real-time gene expression analysis of typical chondrogenic marker genes aggrecan, collagen type II and cartilage oligomeric matrix protein (COMP) as well as of the fibrous marker gene collagen type I. The expression level of marker genes was calculated as percentage of the expression level of the housekeeping gene GAPDH. The bars show the mean (n 5 5) and the SD. Asterisks (*) indicate significance at p < 0.05.

study may point toward the risk of potential hypertrophy and subsequent ossification of the implant in cartilage repair. Therefore, further preclinical animal studies showing cartilage formation without abnormal hypertrophy and ossification are necessary, before TGFB3 can be recommended unrestrictedly for bioactivation of grafts and/or implants in cartilage repair. A limitation of the current and previous studies is that there is no or limited information about the proportion of active TGFB3 in vitro and in vivo over time when given as single dose or continuously by repeated administration. Recently, it has been shown that mesenchymal stem cells seeded in fibrin and/or a collagen type I/III scaffolds as well as scaffolds alone retain the release of TGFB1 and that local or soluble TGFB1 has an impact on the chondrogenic differentiation sequence of mesenchymal stem cells.16 Therefore, further studies are needed that clarify the impact of cell seeding and PGA scaffold material as well as its composition on the release of TGFB3 and thus on the presence of soluble and matrix bound TGFB3 as well as on their impact on mesenchymal progenitor cells’ chondrogenesis.

Continuous application as well as controlled release of bioactive factors or growth factors in vivo or in clinical applications is challenging to realize. Therefore, current clinically applied cartilage repair approaches favor the use of cell-free scaffolds bio-activated by initial or once only addition of bioactive factors during surgery like e.g. the blood derived components PRP14 and serum50,69 or bone marrow aspirates.12,19 However, such patient-derived cocktails of growth factors are undefined, may vary in their growth factor content, and often lack standardization, which may make the outcome of such a procedure hardly predictable. In addition, their use needs further invasive manipulation of the patient for e.g. drawing the blood. Therefore, efforts have been and are made to develop and design carriers for a controlled release of growth factors, in particular TGFB for cartilage repair. Current concepts for carriers facilitating TGFB release comprise the use of microcarriers/microspheres, hydrogels and scaffolds or combinations thereof encapsulating different isoforms of TGFB. For instance, polymer-based microcarriers made of polylactide-polyglycolic acid (PLGA) have been shown to release 70% of TGFB3 over time and to induce cartilage

2572

KRU¨GER et al. b FIGURE 6. Histological analysis of extracellular matrix mineralization by von Kossa staining and gene expression analysis of the chondrocyte hypertrophy marker collagen type X. At day 14, von Kossa staining of mineralized extracellular matrix components was negative in non-stimulated controls (a) and in cultures of progenitor cells in PGA scaffolds with continuous stimulation with 10 ng/mL non-freeze-dried TGFB3 (b) as well as in cultures of progenitor cells embedded in freeze-dried TGFB3-PGA scaffolds (c). Chondrocyte hypertrophy was analyzed by real-time gene expression analysis of collagen type X. The expression level of the marker gene was calculated as percentage of the expression level of the housekeeping gene GAPDH. The bars show the mean (n 5 5) and the SD. Asterisks (*) indicate significance at p < 0.05.

marker molecules like collagen type II and COMP in MSC, in vitro,47 and after implantation of MSC and TGFB3 releasing PLGA microcarriers in SCID mice.6 Hydrogels, self-assembling peptide, agarose and alginate hydrogels retained TGFB1 and its release stimulated chondrogenic differentiation of equine MSC in vitro and of human MSC also after implantation in nude mice.38,55

FIGURE 7. Release of TGFB3 from freeze-dried TGFB3polyglycolic acid (PGA) scaffolds. Release and stability of TGFB3 released from TGFB3-PGA scaffolds was determined during a period of 14 days without removal of the supernatant (a, no medium exchange). To mimic cell culture conditions with regular medium exchange for cell nutrition, TGFB3 released from TGFB3-PGA scaffolds was also determined during a period of 14 days with the first complete exchange of supernatant/medium at day 4 and every other day thereafter as indicated by black arrows (b). The dotted lines represent the mean concentration of TGFB3 found in the supernatants at a given point in time as indicated (n 5 3 per point in time) and SD. The bars (n 5 3 per point in time) show the mean release of TGFB3 from freeze-dried TGFB3-PGA scaffolds between two consecutive points in time (e.g. bar at ‘10 h’ shows TGFB3 released during the time period between ‘6 h’ and ‘10 h’) and SD.

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction

In addition, PLGA scaffolds impregnated with Erk kinase inhibitor PD98059 and immobilized TGFB2 enhanced chondrogenic differentiation of human MSC and repaired osteochondral defects in combination with MSC in the rabbit model.44 Hydrogels such as agarose, fibrin, gelatin, collagen and hyaluronan are frequently used as carrier for growth factor release in cartilage tissue engineering, because of their easy fabrication, controlled delivery of growth factors and inertness.1,10,15,39,61 Other groups favor synthetic polymers to form degradable microspheres for a controlled release combined with good mechanical properties and defined degradation kinetics.34 Aiming at in vivo or clinical application of such growth factor releasing scaffolds for cartilage repair, it has to be considered that in standard in vitro applications, growth factors are renewed for chondrogenic stimulation of cells by simply exchanging the medium every other day. It is obvious that such an approach is not well suited for clinical practice. Therefore, the main advantage and common idea of all these delivery systems is that only a single application of a given growth factor associated with a spatially and temporally controlled release is necessary to achieve chondrogenic differentiation of cells and cartilage repair. Incorporation of growth factors into synthetic polymers by freeze-drying represents a simplified approach, thus leading to a scaffold that allows for local application and rapid release of a given growth factor. Such an approach is in line with our approach using freezedried TGFB3-PGA scaffolds that rapidly release TGFB3 and induce human subchondral mesenchymal progenitor cells toward chondrogenesis. Various factors can influence the release and/or its kinetic of bioactive proteins and have to be considered during manufacturing of bioactive scaffolds for chondrogenic stem cell differentiation. The modification of scaffold geometry and architecture has been reported to have an effect on release kinetics of the bio-molecules.32 Furthermore, encapsulation of TGFB into PLGA microspheres incorporated in hydrogels has been reported to facilitate a better controlled release than microspheres alone. In addition, an initial burst release of TGFB was reduced if microspheres haven been embedded in hydogels.13 The use of additional coating with fibronectin and poly-D-lysine may support stem cell adhesion to pharmacologically active TGFB3-loaded microcarriers made of biodegradable PGLA and polymer 188.47 For a robust application of such systems in cartilage research and repair, it is necessary to achieve a good control over the scaffold properties, the release, the quality of stem cells or chondrocytes as well as stability and activity of bioactive ingredients. Freeze-drying is a standard procedure used in pharmaceutical industry, found its way

2573

into pharmacopoeias and is one of the methods used in the fabrication of biomaterial-based scaffolds.24,25,65 However, a challenge in freeze-drying is reproducibility. Therefore, often optimization of the freeze-drying process and its design in terms of e.g. use of stabilizers, pressure, drying time and temperature is mandatory to achieve high levels of reproducibility and protein stability.48,66 However, in the current study, we achieved a recovery rate of 99.9% for TGFB3 after freeze-drying in PGA scaffolds and subsequent robust chondrogenic differentiation of human subchondral mesenchymal progenitor cells. This may suggest that we designed a robust and reproducible process of freeze-drying bioactivated PGA scaffolds for cartilage repair. In contrast to gels and microcarriers, scaffolds and in particular polymer-based scaffolds like PGA have inherent material characteristics that are favorable in terms of cartilage repair. From the clinical and surgical point of view, PGA-based scaffolds have been proven to support cartilaginous matrix formation in chondrocyte and stem cell based grafts.51,59,68 The PGAbased scaffold has a stable felt-like structure that is flexible and allows for easy cutting to fit the size of the cartilage defect and for secure fixating in the defect by gluing or pin/nail fixation.30,37 However, although the presented in vitro findings are encouraging, future studies, using a relevant animal model, alone or in combination with chondrocytes and/or stem cells, have to show if the TGFB3-PGA scaffold enhances cartilage tissue repair before such an approach can be recommended for clinical application. Therefore, the limitations of the current study are the lack of in vivo data proofing TGFB3-PGA-mediated cartilage repair and the relative short follow-up time that allows evaluating the early phase or onset of progenitor cell differentiation, but not the mid- to long-term effects of initially released TGFB3 on the chondrogenic sequence of subchondral progenitor cells. Although continuous and repeated administration of 10 ng/mL TGFB3 to mesenchymal stem and progenitor cells is the ‘gold standard’ to induce the chondrogenic sequence and although we achieved comparable results by using a freeze-dried TGFB3-PGA scaffold (200 ng/ mL) for progenitor cell chondrogenesis in vitro, further studies about the activity and effective dose of TGFB3 are mandatory to enlighten the underlying mechanisms of TGFB3-mediated chondrogenesis by freeze-dried, bio-activated scaffolds for cartilage repair. In summary, we investigated the potential of a bioactivated freeze-dried TGFB3-polyglycolic acid scaffold to induce chondrogenic differentiation of human subchondral mesenchymal progenitor cells in vitro. The freeze-dried scaffolds facilitated a rapid, initial boost-like release of TGFB3 that was able, without further addition of growth factors, to induce the

KRU¨GER et al.

2574

chondrogenic developmental sequence of human subchondral progenitor cells known from microfracture, accompanied by the induction of typical chondrogenic marker genes including collagen type II and formation of a cartilaginous matrix rich in proteoglycans and collagen type II. These data suggest that bio-activation of polymer-based resorbable scaffolds with TGFB3 using the freeze-drying method is feasible and that such a bio-activated scaffold with its chondrogenesis stimulating properties is a promising tool for stem cellmediated cartilage regeneration.

ACKNOWLEDGMENTS The authors are very grateful to Samuel Vetterlein for the excellent technical assistance. JPK, ME and CK are employees of TransTissue Technologies GmbH. TransTissue develops products on the basis of polymer-scaffolds for the regeneration of mesenchymal tissues. This study was supported by the European Union, EU-FP7 program (TissueGEN: HEALTH-F42011-278955).

REFERENCES 1

Ahmed, T. A., A. Giulivi, M. Griffith, and M. Hincke. Fibrin glues in combination with mesenchymal stem cells to develop a tissue-engineered cartilage substitute. Tissue Eng. Part A 17:323–335, 2011. 2 Alves da Silva, M. L., A. Martins, A. R. Costa-Pinto, V. M. Correlo, P. Sol, M. Bhattacharya, et al. Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan-based scaffolds using a flow-perfusion bioreactor. J. Tissue Eng. Regen. Med. 5:722–732, 2011. 3 Anders, S., M. Volz, H. Frick, and J. Gellissen. A randomized, controlled trial comparing autologous matrixinduced chondrogenesis (AMIC) to microfracture: analysis of 1- and 2-year follow-up data of 2 centers. Open Orthop. J. 7:133–143, 2013. 4 Barry, F., R. E. Boynton, B. Liu, and J. M. Murphy. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp. Cell Res. 268: 189–200, 2001. 5 Bentley, G., L. C. Biant, R. W. Carrington, M. Akmal, A. Goldberg, A. M. Williams, et al. A prospective, randomised comparison of autologous chondrocyte implantation versus mosaicplasty for osteochondral defects in the knee. J. Bone Joint Surg. Br. 85:223–230, 2003. 6 Bouffi, C., O. Thomas, C. Bony, A. Giteau, M. C. VenierJulienne, C. Jorgensen, et al. The role of pharmacologically active microcarriers releasing TGF-beta3 in cartilage formation in vivo by mesenchymal stem cells. Biomaterials 31:6485–6493, 2010. 7 Brittberg, M., A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson, and L. Peterson. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N. Engl. J. Med. 331:889–895, 1994.

8

Cals, F. L., C. A. Hellingman, W. Koevoet, R. J. Baatenburg de Jong, and G. J. van Osch. Effects of transforming growth factor-beta subtypes on in vitro cartilage production and mineralization of human bone marrow stromal-derived mesenchymal stem cells. J. Tissue Eng. Regen. Med. 6:68–76, 2012. 9 Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15:532–534, 1993. 10 Chung, C., and J. A. Burdick. Influence of three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis. Tissue Eng. Part A 15:243–254, 2009. 11 Davies-Tuck, M. L., A. E. Wluka, Y. Wang, A. J. Teichtahl, G. Jones, C. Ding, et al. The natural history of cartilage defects in people with knee osteoarthritis. Osteoarthr. Cartil. 16:337–342, 2008. 12 de Girolamo, L., A. Quaglia, C. Bait, M. Cervellin, E. Prospero, and P. Volpi. Modified autologous matrix-induced chondrogenesis (AMIC) for the treatment of a large osteochondral defect in a varus knee: a case report. Knee Surg. Sports Traumatol. Arthrosc. 20:2287–2290, 2012. 13 DeFail, A. J., C. R. Chu, N. Izzo, and K. G. Marra. Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels. Biomaterials 27:1579–1585, 2006. 14 Dhollander, A. A., F. De Neve, K. F. Almqvist, R. Verdonk, S. Lambrecht, D. Elewaut, et al. Autologous matrix-induced chondrogenesis combined with platelet-rich plasma gel: technical description and a five pilot patients report. Knee Surg. Sports Traumatol. Arthrosc. 19:536–542, 2011. 15 Dickhut, A., V. Dexheimer, K. Martin, R. Lauinger, C. Heisel, and W. Richter. Chondrogenesis of human mesenchymal stem cells by local transforming growth factor-beta delivery in a biphasic resorbable carrier. Tissue Eng. Part A 16:453–464, 2010. 16 Diederichs, S., K. Baral, M. Tanner, and W. Richter. Interplay between local versus soluble transforming growth factor-beta and fibrin scaffolds: role of cells and impact on human mesenchymal stem cell chondrogenesis. Tissue Eng. Part A 18:1140–1150, 2012. 17 Endres, M., K. Neumann, T. Haupl, C. Erggelet, J. Ringe, M. Sittinger, et al. Synovial fluid recruits human mesenchymal progenitors from subchondral spongious bone marrow. J. Orthop. Res. 25:1299–1307, 2007. 18 Endres, M., K. Neumann, B. Zhou, U. Freymann, D. Pretzel, M. Stoffel, et al. An ovine in vitro model for chondrocyte-based scaffold-assisted cartilage grafts. J Orthop. Surg. Res. 7:37, 2012. 19 Enea, D., S. Cecconi, S. Calcagno, A. Busilacchi, S. Manzotti, C. Kaps, et al. Single-stage cartilage repair in the knee with microfracture covered with a resorbable polymer-based matrix and autologous bone marrow concentrate. Knee 20:562–569, 2013. 20 Erggelet, C., M. Endres, K. Neumann, L. Morawietz, J. Ringe, K. Haberstroh, et al. Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell-free polymer-based implants. J. Orthop. Res. 27:1353–1360, 2009. 21 Erggelet, C., K. Neumann, M. Endres, K. Haberstroh, M. Sittinger, and C. Kaps. Regeneration of ovine articular cartilage defects by cell-free polymer-based implants. Biomaterials 28:5570–5580, 2007. 22 Fan, H., C. Zhang, J. Li, L. Bi, L. Qin, H. Wu, et al. Gelatin microspheres containing TGF-beta3 enhance the

Freeze-Dried Bioactive Polymer-Based Scaffolds for Chondrogenic Induction chondrogenesis of mesenchymal stem cells in modified pellet culture. Biomacromolecules 9:927–934, 2008. 23 Frisbie, D. D., J. T. Oxford, L. Southwood, G. W. Trotter, W. G. Rodkey, J. R. Steadman, et al. Early events in cartilage repair after subchondral bone microfracture. Clin. Orthop. Relat. Res. 407:215–227, 2003. 24 Garg, T., and A. K. Goyal. Biomaterial-based scaffolds—current status and future directions. Expert Opin. Drug Deliv. 11:767–789, 2014. 25 Garg, T., O. Singh, S. Arora, and R. Murthy. Scaffold: a novel carrier for cell and drug delivery. Crit. Rev. Ther. Drug Carr. Syst. 29:1–63, 2012. 26 Gille, J., E. Schuseil, J. Wimmer, J. Gellissen, A. P. Schulz, and P. Behrens. Mid-term results of autologous matrixinduced chondrogenesis for treatment of focal cartilage defects in the knee. Knee Surg. Sports Traumatol. Arthrosc. 18:1456–1464, 2010. 27 Goyal, D., S. Keyhani, E. H. Lee, and J. H. Hui. Evidencebased status of microfracture technique: a systematic review of level I and II studies. Arthroscopy 29:1579–1588, 2013. 28 Gudas, R., R. J. Kalesinskas, V. Kimtys, E. Stankevicius, V. Toliusis, G. Bernotavicius, et al. A prospective randomized clinical study of mosaic osteochondral autologous transplantation versus microfracture for the treatment of osteochondral defects in the knee joint in young athletes. Arthroscopy 21:1066–1075, 2005. 29 Hegewald, A. A., J. Ringe, J. Bartel, I. Kruger, M. Notter, D. Barnewitz, et al. Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study. Tissue Cell 36:431–438, 2004. 30 Herbort, M., S. Zelle, D. Rosenbaum, N. Osada, M. Raschke, W. Petersen, et al. Arthroscopic fixation of matrix-associated autologous chondrocyte implantation: importance of fixation pin angle on joint compression forces. Arthroscopy 27:809–816, 2011. 31 Horas, U., R. Schnettler, D. Pelinkovic, G. Herr, and T. Aigner. Osteochondral transplantation versus autogenous chondrocyte transplantation. A prospective comparative clinical study. Chirurg 71:1090–1097, 2000. 32 Huang, C. L., W. L. Lee, and J. S. Loo. Drug-eluting scaffolds for bone and cartilage regeneration. Drug Discov. Today 19:714–724, 2014. 33 Hunziker, E. B., I. M. Driesang, and E. A. Morris. Chondrogenesis in cartilage repair is induced by members of the transforming growth factor-beta superfamily. Clin. Orthop. Relat. Res. 391:S171–S181, 2001. 34 Jaklenec, A., A. Hinckfuss, B. Bilgen, D. M. Ciombor, R. Aaron, and E. Mathiowitz. Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds. Biomaterials 29:1518–1525, 2008. 35 Johnstone, B., T. M. Hering, A. I. Caplan, V. M. Goldberg, and J. U. Yoo. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238:265–272, 1998. 36 Kang, S. W., L. P. Bada, C. S. Kang, J. S. Lee, C. H. Kim, J. H. Park, et al. Articular cartilage regeneration with microfracture and hyaluronic acid. Biotechnol. Lett. 30: 435–439, 2008. 37 Knecht, S., C. Erggelet, M. Endres, M. Sittinger, C. Kaps, and E. Stussi. Mechanical testing of fixation techniques for scaffold-based tissue-engineered grafts. J. Biomed. Mater. Res. B 83B:50–57, 2007. 38 Kopesky, P. W., S. Byun, E. J. Vanderploeg, J. D. Kisiday, D. D. Frisbie, and A. J. Grodzinsky. Sustained delivery of

2575

bioactive TGF-beta1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells. J. Biomed. Mater. Res. A 102:1275–1285, 2014. 39 Kopesky, P. W., E. J. Vanderploeg, J. D. Kisiday, D. D. Frisbie, J. D. Sandy, and A. J. Grodzinsky. Controlled delivery of transforming growth factor beta1 by selfassembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2/3 signaling. Tissue Eng. Part A 17:83–92, 2011. 40 Kreuz, P. C., C. Erggelet, M. R. Steinwachs, S. J. Krause, A. Lahm, P. Niemeyer, et al. Is microfracture of chondral defects in the knee associated with different results in patients aged 40 years or younger? Arthroscopy 22:1180– 1186, 2006. 41 Kreuz, P. C., M. R. Steinwachs, C. Erggelet, S. J. Krause, G. Konrad, M. Uhl, et al. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthr. Cartil. 14:1119–1125, 2006. 42 Kruger, J. P., U. Freymann, S. Vetterlein, K. Neumann, M. Endres, and C. Kaps. Bioactive factors in platelet-rich plasma obtained by apheresis. Transfus. Med. Hemother. 40:432–440, 2013. 43 Kruger, J. P., S. Hondke, M. Endres, A. Pruss, A. Siclari, and C. Kaps. Human platelet-rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells. J. Orthop. Res. 30:845–852, 2012. 44 Lee, J. M., J. D. Kim, E. J. Oh, S. H. Oh, J. H. Lee, and G. I. Im. PD98059-impregnated functional PLGA scaffold for direct tissue engineering promotes chondrogenesis and prevents hypertrophy from mesenchymal stem cells. Tissue Eng. Part A 20:982–991, 2014. 45 Li, W. J., R. Tuli, X. Huang, P. Laquerriere, and R. S. Tuan. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 26:5158–5166, 2005. 46 Miyanishi, K., M. C. Trindade, D. P. Lindsey, G. S. Beaupre, D. R. Carter, S. B. Goodman, et al. Effects of hydrostatic pressure and transforming growth factor-beta 3 on adult human mesenchymal stem cell chondrogenesis in vitro. Tissue Eng. 12:1419–1428, 2006. 47 Morille, M., T. Van-Thanh, X. Garric, J. Cayon, J. Coudane, D. Noel, et al. New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release 170:99–110, 2013. 48 Nail, S. L., S. Jiang, S. Chongprasert, and S. A. Knopp. Fundamentals of freeze-drying. Pharm. Biotechnol. 14: 281–360, 2002. 49 Neumann, K., T. Dehne, M. Endres, C. Erggelet, C. Kaps, J. Ringe, et al. Chondrogenic differentiation capacity of human mesenchymal progenitor cells derived from subchondral cortico-spongious bone. J. Orthop. Res. 26: 1449–1456, 2008. 50 Patrascu, J. M., U. Freymann, C. Kaps, and D. V. Poenaru. Repair of a post-traumatic cartilage defect with a cell-free polymer-based cartilage implant: a follow-up at two years by MRI and histological review. J. Bone Joint Surg. Br. 92:1160–1163, 2010. 51 Patrascu, J. M., J. P. Kruger, H. G. Boss, A. K. Ketzmar, U. Freymann, M. Sittinger, et al. Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model. J. Biomed. Mater. Res. B 101:1310–1320, 2013.

2576 52

KRU¨GER et al.

Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, et al. Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147, 1999. 53 Ravindran, S., J. L. Roam, P. K. Nguyen, T. M. Hering, D. L. Elbert, and A. McAlinden. Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-beta3. Biomaterials 32:8436–8445, 2011. 54 Redler, L. H., S. A. Thompson, S. H. Hsu, C. S. Ahmad, and W. N. Levine. Platelet-rich plasma therapy: a systematic literature review and evidence for clinical use. Phys. Sportsmed. 39:42–51, 2011. 55 Re’em, T., Y. Kaminer-Israeli, E. Ruvinov, and S. Cohen. Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds. Biomaterials 33:751–761, 2012. 56 Schagemann, J. C., S. Paul, M. E. Casper, J. Rohwedel, J. Kramer, C. Kaps, et al. Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-epsilon-caprolactone scaffolds. J. Biomed. Mater. Res. A 101:1620–1628, 2013. 57 Schmitt, B., J. Ringe, T. Haupl, M. Notter, R. Manz, G. R. Burmester, et al. BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. Differentiation 71:567–577, 2003. 58 Shapiro, F., S. Koide, and M. J. Glimcher. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 75:532–553, 1993. 59 Siclari, A., G. Mascaro, C. Gentili, R. Cancedda, and E. Boux. A cell-free scaffold-based cartilage repair provides improved function hyaline-like repair at one year. Clin. Orthop. Relat. Res. 470:910–919, 2012. 60 Siclari, A., G. Mascaro, C. Gentili, C. Kaps, R. Cancedda, and E. Boux. Cartilage repair in the knee with subchondral drilling augmented with a platelet-rich plasma-immersed polymer-based implant. Knee Surg. Sports Traumatol. Arthrosc. 22:1225–1234, 2014.

61

Solorio, L. D., C. D. Dhami, P. N. Dang, E. L. Vieregge, and E. Alsberg. Spatiotemporal regulation of chondrogenic differentiation with controlled delivery of transforming growth factor-beta1 from gelatin microspheres in mesenchymal stem cell aggregates. Stem Cells Transl. Med. 1:632–639, 2012. 62 Stanish, W. D., R. McCormack, F. Forriol, N. Mohtadi, S. Pelet, J. Desnoyers, et al. Novel scaffold-based BST-CarGel treatment results in superior cartilage repair compared with microfracture in a randomized controlled trial. J. Bone Joint Surg. Am. 95:1640–1650, 2013. 63 Steadman, J. R., W. G. Rodkey, K. K. Briggs, and J. J. Rodrigo. The microfracture technic in the management of complete cartilage defects in the knee joint. Orthopade 28:26–32, 1999. 64 Steadman, J. R., W. G. Rodkey, and J. J. Rodrigo. Microfracture: surgical technique and rehabilitation to treat chondral defects. Clin. Orthop. Relat. Res. 391:S362– S369, 2001. 65 Stickings, P., P. Rigsby, K. H. Buchheit, and D. Sesardic. Calibration of European pharmacopoeia biological reference preparation for diphtheria vaccine (adsorbed) batch 4. Pharmeur. Bio. Sci. Notes 1–9:2009, 2009. 66 Tang, X., and M. J. Pikal. Design of freeze-drying processes for pharmaceuticals: practical advice. Pharm. Res. 21:191–200, 2004. 67 Tang, Q. O., K. Shakib, M. Heliotis, E. Tsiridis, A. Mantalaris, and U. Ripamonti. TGF-beta3: a potential biological therapy for enhancing chondrogenesis. Expert Opin. Biol. Ther. 9:689–701, 2009. 68 Trimborn, M., M. Endres, C. Bommer, U. Janke, J. P. Kruger, L. Morawietz, et al. Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering. Acta Biomater. 8:1519–1529, 2012. 69 Zantop, T., and W. Petersen. Arthroscopic implantation of a matrix to cover large chondral defect during microfracture. Arthroscopy 25:1354–1360, 2009.

Initial boost release of transforming growth factor-β3 and chondrogenesis by freeze-dried bioactive polymer scaffolds.

In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to...
2MB Sizes 1 Downloads 4 Views