BMP activation and Wnt-signalling affect biochemistry and functional biomechanical properties of cartilage tissue engineering constructs.

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YJOCA3028_proof ■ 2 December 2013 ■ 1/9

Osteoarthritis and Cartilage xxx (2013) 1e9

BMP activation and Wnt-signalling affect biochemistry and functional biomechanical properties of cartilage tissue engineering constructs Q6

A. Krase y, R. Abedian z, E. Steck y, C. Hurschler z, W. Richter y * y Research Center for Experimental Orthopaedics, Department of Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Heidelberg, Germany z Laboratory for Biomechanics and Biomaterials, Orthopaedic Department, Hannover Medical School of Hannover, Hannover, Germany

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a r t i c l e i n f o

s u m m a r y

Article history: Received 28 August 2013 Accepted 15 November 2013

Objectives: Bone morphogenetic protein (BMP-) and Wnt-signalling play crucial roles in cartilage homeostasis. Our objective was to investigate whether activation of the BMP-pathway or stimulation of Wnt-signalling cascades effectively enhances cartilage-specific extracellular matrix (ECM) accumulation and functional biomechanical parameters of chondrocyte-seeded tissue engineering (TE)-constructs. Design: Articular chondrocytes were cultured in collagen-type-I/III-matrices over 6 weeks to create a biomechanical standard curve. Effects of stimulation with 100 ng/mL BMP-4/-7 heterodimer or 10 mM lithium chloride (LiCl) on ECM-deposition was quantified and characterized histologically. Biomechanical parameters were determined by the Very Low Rubber Hardness (VLRH) method and under confined compression stress relaxation. Results: BMP-4/-7 treatment resulted in stronger collagen type-II staining and significantly enhanced glycosaminoglycan (GAG) deposition (3.2-fold; *P < 0.01) correlating with improved hardness (w1.7fold; *P ¼ 0.001) reaching 83% of native cartilage values after 28 days, a value not reached before 9 weeks without stimulation. LiCl treatment enhanced VLRH slightly, but significantly (w1.3-fold; *P ¼ 0.016) with a trend to more ECM-deposition. BMP-4/-7 treatment significantly enhanced the E Modulus (105.7 34.1 kPa; *P ¼ 0.000001) compared to controls (8.0 4.2 kPa). Poisson’s ratio was significantly improved by BMP-4/-7 treatment (0.0703 0.0409; *P ¼ 0.013) vs controls (0.0432 0.0284) and a significantly lower permeability (5.8 2.1056 1014 m^4/N.s; *P ¼ 0.00001) was detected compared to untreated scaffolds (4.4 3.1289 1013 m^4/N.s). Conclusions: While Wnt-activation is less effective, BMP-4/-7 heterodimer stimulation approximated native cartilage features in less than 50% of standard culture time representing a promising strategy for functional cartilage TE to improve biomechanical parameters of engineered cartilage. Ó 2013 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Keywords: Cartilage tissue engineering Extracellular matrix proteins Biomechanical properties BMP-4/-7 heterodimer Lithium chloride

Introduction As a highly specialized tissue, articular cartilage protects underlying bone from destructive forces mainly caused by the specific composition of the extracellular matrix (ECM), which consists of a highly ordered framework of several types of collagens and proteoglycan1. Tensile strength of the tissue is attributed predominantly to collagen type II, while proteoglycans contribute mainly to * Address correspondence and reprint requests to: W. Richter, Research Center for Experimental Orthopaedics, Department of Orthopaedics, Trauma Surgery and Paraplegiology, Heidelberg University Hospital, Schlierbacher Landstraße 200a, 69118 Heidelberg, Germany. Tel: 49-(0)-6221-562-9253; Fax: 49-(0)-6221-5629288. E-mail addresses: [email protected] (A. Krase), Abediyan@ gmail.com (R. Abedian), [email protected] (E. Steck), [email protected] (C. Hurschler), [email protected] (W. Richter).

compressive properties in two ways: a bulk of proteoglycan aggregates are immobilized within the collagen fibre network and, associated with the negative charge of the molecules, proteoglycans bind water which leads to a swelling-pressure2. Damage of native tissue as consequence of trauma or due to a degenerative joint disease such as osteoarthritis (OA)3, is related to a loss of the healthy ECM structure that generally leads to a deficit in functional biomechanical characteristics. In particular, in chondral defects, where the lesion is located within articular cartilage, progenitor cells from blood and bone marrow cannot enter the damaged region to contribute to repair owing to a lack of vascularization. Additionally, resident chondrocytes are unable to migrate into the defect and, therefore, the lesion is not filled with repair tissue and remains permanently4,5. To overcome this problem, tissue engineering (TE) approaches based on carrier materials seeded with chondrocytes are currently under investigation in order to enhance

1063-4584/$ e see front matter Ó 2013 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. http://dx.doi.org/10.1016/j.joca.2013.11.011

Please cite this article in press as: Krase A, et al., BMP activation and Wnt-signalling affect biochemistry and functional biomechanical properties of cartilage tissue engineering constructs, Osteoarthritis and Cartilage (2013), http://dx.doi.org/10.1016/j.joca.2013.11.011

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A. Krase et al. / Osteoarthritis and Cartilage xxx (2013) 1e9

biochemical and biomechanical characteristics of in vitro engineered cartilage. Improved biomechanical properties of TE-constructs were achieved by increasing the ECM-deposition as a result of a prolonged culture period6e8, enhanced cell seeding density7, physical9 or mechanical stimulation during culture7,10,11. Additionally, the combination of growth factor treatment and dynamic loading in chondrocyte-seeded TE-constructs revealed a synergistic effect on functional properties12. However, the unique functional biomechanical characteristics of articular cartilage still have to be reached6,13. A number of biological factors affecting important signalling pathways of cartilage ECM-deposition have been shown to enhance functional properties of chondrocyte-seeded TE-constructs. Members of the transforming growth factor (TGF)-b superfamily such as bone morphogenetic protein (BMP) homodimers are promising inducers of cartilage matrix synthesis14,15. Interestingly, heterodimeric BMP ligands have been demonstrated to display a higher activity compared to their homodimeric counterparts16. While homodimers form symmetric molecules binding to a restricted set of BMP type I-receptors with high affinity, BMP heterodimers can form asymmetric ligands with broader BMP-receptor binding resulting in enhanced signalling activity17,18. Of the BMP homodimers, BMP-4 and BMP-7 have been identified as potent stimulators of articular chondrocyte anabolism in monolayer and 3D culture19e21. Additionally, BMP-4 and BMP-7 target distinct BMP type I-receptors: while BMP-4 possesses a high affinity to activinlike-kinase (ALK)-3 and ALK-6 receptors, BMP-7 preferentially binds ALK-222,23. Stimulation of ectopic bone formation with BMP4/-7 heterodimer in rats resulted in superior bone formation in comparison with BMP homodimer24. In line, murine embryonic fibroblasts, overexpressing either BMP-2, BMP-4, BMP-7 homodimer, respectively, or the combination of two different BMPs before ectopic transplantation into mice, revealed that the combination of BMP-4/-7 resulted in two- to threefold more bone25. The activity of BMP heterodimers on cartilage ECM-production by chondrocytes remained so far, however, enigmatic. Another essential pathway involved in chondrogenesis and joint formation is the Wnt-signalling cascade26,27, which regulates chondrocyte proliferation, differentiation and maintenance of the cell phenotype28,29. Canonical Wnt-signalling is mediated by bcatenin which in the presence of lithium chloride (LiCl) accumulates in the cytoplasm and then translocates into the nucleus. There, b-catenin forms a complex with transcription factors to activate the transcription of target genes. Stimulation of Wnt-signalling by means of the Wnt agonist LiCl in an undifferentiated mesenchymal stem cell line stimulated expression of chondrogenic marker genes like COL2 and aggrecan30. Others demonstrated elevated proteoglycan levels after LiCl treatment of mesenchymal stromal cells during chondrogenic differentiation31. Nevertheless it is still unclear whether Wnt-activation affects ECM-deposition by chondrocytes in a TE-matrix. A variety of methods to describe functional properties of cartilage and TE-constructs have been identified. Indentation32,33 and confined compression34e37 are established methods to study mechanical properties of tissue due to the simplicity of loading configurations and the ability to achieve the desired boundary conditions. We have furthermore recently identified a new sensitive method to evaluate mechanical properties of in vitro generated cartilage constructs38. This indentation test, following the Very Low Rubber Hardness (VLRH) principle, was originally designed to analyse soft materials like silicone39 and may represent a sensitive system detecting consequences of ECM stimulation in cartilage TEconstructs in terms of absolute hardness values. While BMP- and Wnt-signalling act through well-studied pathways, surprisingly, it is still not known whether their activation either by a BMP heterodimer or by LiCl, respectively, affects

biomechanical properties of chondrocyte-seeded articular cartilage TE-constructs. Therefore, the objective of this study was to analyse whether activation of BMP- or Wnt-signalling is effective to enhance biomechanical properties of chondrocyte-seeded collagen matrix-based TE-constructs holding potential to further improve therapeutic strategies for cartilage repair. Articular chondrocytes were cultured in a collagen carrier in the presence of BMP-4/-7 heterodimer or the Wnt agonist LiCl in chondrogenic medium for 4e6 weeks. Following factor treatment, a detailed characterization of biochemical and biomechanical parameters of TE-constructs was performed relating to the properties of control cultures and native cartilage tissue. Materials and methods Cell isolation and cultivation Articular chondrocytes were isolated from porcine knee joints (n ¼ 5 donors). Rinsed cartilage was cut into small pieces, digested overnight with collagenase B (Roche) and hyaluronidase (Sigmae Aldrich), as described14,40. Chondrocytes were filtered through a 50 mm nylon mesh washed in PBS and seeded at 8500 cells/cm2. Chondrocytes were expanded for two to three population doublings (PD) in DMEM (low-glucose) with 10% foetal calf serum (FCS) (Biochrom), 10 U/mL penicillin, 100 mg/mL streptomycin (Biochrom) at 37 C and 6% CO2. Medium was replaced twice a week. Cartilage TE-constructs Chondrocytes were harvested and suspended in chondrogenic medium (high-glucose DMEM with 0.1 mM dexamethasone, 0.17 mM ascorbic acid-2 phosphate, 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL sodium selenite, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/mL bovine serum albumin (BSA), 100 U/ mL penicillin, 100 mg/mL streptomycin) with 10 ng/mL TGFb-1 (Miltenyi Biotech) at 5 107 cells/mL. Collagen type I/III matrices (Ø 5 mm, 1.5 mm height; Matricel) were seeded with 30 mL cell suspension following the manufacturer’s drop-on protocol (1.5 106 cells/construct). Cells were allowed to attach for 2 h until 2 mL of chondrogenic medium or chondrogenic medium containing 100 ng/mL BMP-4/-7 (R & D Systems Inc.) or 10 mM LiCl (SigmaeAldrich) were added. Five constructs per donor and group were cultured in independent experiments for 0, 7, 21, 28 and 42 days with medium changed three times a week. Thickness at termination of culture was measured at the centre point by a needle probe method41. A custom MATLABÒ procedure (MATLABÒ, Linux version 2011b, Mathworks Inc.) was used to evaluate the results. Histology Samples were fixed in 1 mL 4% paraformaldehyde dehydrated using a graded alcohol series and embedded in paraffin. For the detection of proteoglycans, 5 mm sections were stained with safranin O (0.2% in 1% acetic acid, Chroma) and Certistain fast green (0.04% in 0.2% acetic acid, Merck) using a standard procedure. Immunohistology for collagen type II was performed as described42. Briefly, deparaffinised and rehydrated sections were treated with 4 mg/mL hyaluronidase in PBS pH 5.5 (SigmaeAldrich) and 1 mg/mL pronase (Roche). Non-specific background was blocked with 5% BSA and sections were incubated with anti-human collagen type II antibody (1:1000, clone II-4C11, ICN Biomedicals) followed by biotinylated goat anti-mouse antibody (1:500, Dianova), and streptavidinealkaline phosphatase (Dako) and fast red detection (SigmaeAldrich).


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Biochemical analysis After 0, 7, 21, 28 and 42 days of chondrogenic culture total glycosaminoglycan (GAG-) content was analysed individually in two constructs per donor (n ¼ 5 donors) unless indicated otherwise. Freeze-dried constructs were digested with 0.5 mg/mL Proteinase K (Roche) at 60 C and 800 rpm for 16 h. Aliquots were analysed for GAG-content using 1,9-dimethyl-methylene-blue (DMMB) assay43. Values were normalized to a chondroitin-6-sulphate standard curve. Digests were analysed for their DNA-content by QuantiTÔPicoGreenÒ dsDNA Assay Kit (Life Technologies) according to the manufactures instructions. Gene expression analysis Constructs were minced, and totalRNA was isolated by a phenol/ guanidine isothiocyanate extraction reagent (peqGOLD TriFast, PEQLAB). Polyadenylated mRNA was purified from totalRNA with oligo(dT)-coupled magnetic beads (Dynabeads, Life Technologies). After complementary DNA (cDNA) synthesis using Omniscript reverse transcriptase (Qiagen) and oligo(dT) primers. qRT-PCR was performed on LightCyclerÒ 96 (Roche) with the following primers: RPL13A 50 -ACTGAAGTACCTGGCCTTCC-30 and 50 -CATCGAA0 0 CACCTTGAGACG-3 ; COL2A1 5 -CACGGATGGTCCCAAAGG-30 and 50 -ATACCAGCAGCTCCCCTCT-30 ; AXIN2 50 -GGTTTGCCTGCAATGGATTC-3ànd 50 -CTGTGTTTTCTCCCCCACTC-30 . Biomechanical testing Unconfined indentation testing was applied individually directly after culture (five constructs/donor; five donors) on a titanium plate using the mechanical testing device Digi-Test-II, with a spherical 2.5 mm-diameter stamp (DTVLRH 6995) and Hardtest 2.0 software (Bareiss). Mechanical testing defined by DIN ISO 27588 following the VLRH principle was applied. Testing of each scaffold generated 600 successive relative hardness values (VLRH) over 600 ms, characterizing the loading (1e300 ms) and recovery properties (301e 600 ms) of the evaluated scaffolds over time. The calculation of VLRH values based upon the depth of indentation (D in mm) of the stamp under the constant loading force of 100 mN. The linear relationship between both parameters is described as follows39:

VLRH ¼ 100 0:1D

(1)

Hardness values ranged from 0 to 100. Each sample was analysed in duplicate. Biomechanical testing under confined compression stress relaxation was applied after 42 days in culture. Measurement was performed in a stainless steel cylindrical fixture of the same diameter as the specimens to constrain lateral deformation assuring the confined compression boundary conditions. In a ZwickÒ commercial testing machine (Zwick) uniaxial compressive loading was applied by means of a glass porous plunger of the same diameter as the specimens to allow the fluid exchange to and from the cartilage outer surface. A stress relaxation protocol was used in which the specimens were compressed at a displacement rate of 0.15 mm/s to a displacement step of 0.5 mm i.e., 25e30% of axial strain depending on the specimen thickness, and held at that deformation until an equilibrium stress was attained (37 C in 0.15 M PBS). A total of 55 scaffolds were measured yielding in 55 confined compression data points. To evaluate the results of stress relaxation confined compression tests a custom Finite-Element (FE)-based optimized parameter extraction routine was applied to derive the material parameters of a biphasic poroelastic material model. For the material representation,

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a solid matrix with hyperelastic compressible neo-Hookean properties and an inviscid fluid phase were used, which introduce three material parameters: E modulus, Poisson’s ratio and hydraulic permeability44. The neo-Hookean material is an extension of Hooke’s law for the case of large deformations. It is appropriate for certain plastics and rubber-like substances45. FE models were generated to match the individual dimensions of each specimen, and a cost-function was defined as the sum of squares of the difference between individual points of experimental and simulated creep deformation curves. An optimization routine was written based on a Simplex algorithm using the fminsearch function from the function from the MATLABÒ library, and the parameter-set of the FE simulations optimized until the calculated time-dependent stress relaxation curve most closely matched the experiment results (FEBioÒ, Linux version 1.3). The complete optimization and FE modelling process was conducted on a Linux machine with Ubuntu 12.4. For the statistical analysis results were grouped together into three clusters: control (n ¼ 20), BMP-4/-7 (n ¼ 20), and LiCl (n ¼ 15). Statistical analysis Collection of data was performed individually for each donor. Each construct was processed independently and data of one donor were summarized. Data of time points or treatment groups were grouped together, followed by ManneWhitney U test (non-paired analyses) and Bonferroni post-hoc. Data were analysed with SPSS for Windows 19.0 (SPSS Inc.) and processed with MS Excel 2010 (Microsoft). Graphs show either mean 95% confidence interval or data are presented as BoxeWhisker plot. Each box represents the interquartile range (IQR) extending between the 25th and 75th percentiles. Lines inside the boxes represent the median. Biomechanical properties were correlated with quantitative GAG-content using a two-tailed Pearson’s correlation with a significance level of 0.05. Results Time-dependent deposition of ECM allows generation of a biomechanical standard curve The collagen type I/III scaffold provided a directed parallel 3D fibre structure and allowed cartilage-like ECM-deposition under chondrogenic standard conditions in vitro. Scaffolds maintained a cylindrical shape and a whitish glossy cartilage-like surface developed (data not shown). Histological examination showed a positive signal for collagen type II and safranin O [Fig. 1(A)], increasing over time with strong staining at day 42. Dry weight of constructs steadily rose over the examination period and reached significantly higher levels at day 42 (3.4 mg per scaffold; *P ¼ 0.032) compared to day 0 [Fig. 1(B)]. GAG-deposition increased during culture reaching significantly higher levels at day 42 (724.1 378.9 mg GAG/scaffold; *P ¼ 0.032) compared to day 0 (45.9 4.3 mg) [Fig. 1(C)]. Total DNA-amount did not change significantly. At day 42 about 90% of the day 0 DNA-content was detected (data not shown). Hardness values of chondrocyte-seeded scaffolds were determined at day 0, 7, 21 and 42 by indentation testing. Over the examination period, relative hardness increased in a time-dependent manner [Fig. 1(D)]. Under closer scrutiny, absolute hardness values at the last loading point (300 ms) indicated cumulative biomechanical properties over time. Biomechanical hardness on day 0 (5.7 1.1) increased significantly until day 7 (37.6 12.0; *P ¼ 0.001). A significant increase was observed between all following time points, reaching highest values at day 42 (60.7 7.1;


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Fig. 1. ECM-deposition, dry weight and absolute hardness of chondrocyte-seeded scaffolds over time. Collagen sponges were seeded with 1.5 106 porcine chondrocytes of five donors in five individual experiments and cultured in chondrogenic medium over the indicated time span. (A) (Immuno-)Histological examination of the deposition of collagen type II and proteoglycan (n ¼ 10 constructs per time point). Representative pictures of constructs from one donor are shown. (B) Scaffolds harvested at indicated time points were freezedried and weighted (n ¼ 10 constructs per time point). ManneWhitney U test *P ¼ 0.032 compared to day 0. Values are presented as mean 95% confidence interval. (C) Quantification of the total GAG-content per scaffold over time (n ¼ 10 constructs per time point). ManneWhitney U test *P ¼ 0.032 compared to day 0. Values are the mean 95% confidence interval. (D) Evaluation of absolute hardness. Samples were subjected to unconfined biomechanical indentation testing by the Bareiss mechanical testing device DigiTest-II. Testing of each construct generated 600 relative hardness values over 600 ms (hardness range 0e100 according to VLRH DIN norm ISO 27588) characterizing the loading phase (1e300 ms) and recovery properties (301e600 ms) of the evaluated scaffold. Mean values of n ¼ 25 scaffolds (five constructs per donor, five donors) are shown per time point. (E) Evaluation of absolute hardness values at the last loading point (300 ms) (n ¼ 25 constructs per time point). ManneWhitney U test, Bonferroni post-hoc. Values are presented as Q5 mean 95% confidence interval. d0ed7 #P ¼ 0.001; d7ed21 #P ¼ 0.000305; d21ed42 #P ¼ 0.00062.

*P ¼ 0.000062) [Fig. 1(E)]. Thus, significant differences between succeeding time points allowed generation of a biomechanical standard curve. Indentation testing of native articular cartilage discs from porcine knees (n ¼ 3) revealed a mean VLRH (300 ms) of 88.9 2.4. Thus, in vitro TE-constructs cultured for 6 weeks reached about 68% of values of native cartilage emphasizing a need for further stimulation of adequate ECM-deposition in shorter culture periods. BMP-4/-7- but not Wnt-activation stimulates ECM-deposition

is

To identify whether activation of BMP- or Wnt-signalling effective to enhance ECM-deposition of TE-constructs,

consequences of BMP-4/-7 heterodimer or LiCl treatment were analysed. According to histological evaluation of collagen type II and proteoglycan, BMP-4/-7 treated constructs contained increased cartilage-like ECM [Fig. 2(A)]. While BMP- and Wnt-signalling pathway are involved in hypertrophic differentiation of chondrocytes, it is noteworthy that cell activation did not initiate the deposition of collagen type X, according to histology (n ¼ 2 per group; data not shown). Total GAG-content per scaffold was significantly higher after treatment with 100 ng/ml BMP-4/-7 (1219.4 406.4 mg; 3.2-fold; *P < 0.01) compared to controls (379.9 77.4 mg). In contrast, LiCl treatment revealed only a trend to higher GAG-deposition reaching no significant effect (630.0 122.0 mg GAG/scaffold; not shown). At


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Fig. 2. Effect of BMP-4/-7 and LiCl treatment on chondrocyte-seeded TE-constructs. After seeding collagen sponges with 1.5 106 porcine chondrocytes, scaffolds were cultivated under chondrogenic conditions (control) or stimulated with 100 ng/mL BMP-4/-7 heterodimer (BMP-4/-7) and 10 mM LiCl for 4 weeks, respectively. Cells of three donors were examined individually. (A) Histological detection of proteoglycan deposition by safranin O staining. Insets show collagen type II immunohistochemistry. One representative construct per group is shown. White arrows indicate parallel collagen fibre orientation of the scaffold. (B) Quantification of mg GAG per mg DNA. A total of six scaffolds per group were analysed. ManneWhitney U test, Bonferroni post-hoc. Control vs BMP-4/-7 *P ¼ 0.01; BMP-4/-7 vs LiCl *P ¼ 0.022. Data are presented as BoxeWhisker plots. Each box represents the IQR extending between the 25th and 75th percentiles. Lines inside the boxes represent the median of six independent scaffolds. (C) Determination of thickness by needle probe at day 42. Measurement of 15e20 samples per group. ManneWhitney U test, Bonferroni post-hoc. Control vs BMP-4/-7 *P ¼ 0.000006; BMP-4/-7 vs LiCl *P ¼ 0.000002. Data are presented as BoxeWhisker plots. (D) mRNA expression levels of COL2A1 relative to the reference gene RPL13A. A total of six scaffolds were analysed. Manne Whitney U test, Bonferroni post-hoc. Control vs BMP-4/-7 *P ¼ 0.000033; BMP-4/-7 vs LiCl *P ¼ 0.003. Data are presented as BoxeWhisker plots. (E) mRNA expression levels of AXIN2 relative to the reference gene RPL13A. A total of six scaffolds were analysed. ManneWhitney U test, Bonferroni post-hoc. Control vs LiCl *P ¼ 0.04; BMP-4/-7 vs LiCl *P ¼ 0.045. Data are presented as BoxeWhisker plots.

day 28, scaffolds of the BMP-4/-7 and the LiCl group contained more cells compared to controls according to a significantly elevated DNA-content (data not shown). GAG-content referred to mg demonstrated that cells of the BMP-4/-7 group deposited significantly more GAG per cell (1264.5 343.3; 1.8-fold; *P ¼ 0.01) compared to controls (718.4 152.3). Again, LiCl treatment revealed only a trend to higher GAG-deposition per cell reaching no significant effect (910.8 147.5) [Fig. 2(B)]. As a result, BMP-4/-7stimulated constructs, but not those treated with LiCl, grew significantly thicker than control scaffolds [Fig. 2(C)]. To assess whether BMP-4/-7 affected gene expression of COL2A1 and the Wnt-pathway-regulated AXIN2 gene, mRNA levels were quantified in independent scaffolds (n ¼ 6, two from three donors). Analysis revealed a significant increase of COL2A1 expression after treatment with BMP-4/-7 (*P ¼ 0.000033) but not after treatment with LiCl [Fig. 2(D)] while AXIN2 mRNA expression was only stimulated in the LiCl group (*P ¼ 0.04) [Fig. 2(E)].

BMP-4/-7 stimulation improves biomechanical function Biomechanical characterization of scaffolds revealed that enhanced ECM-deposition in BMP-4/-7 treated samples positively affected their functional performance. Hardness was significantly enhanced, reaching absolute values of 73.5 6.9 (*P ¼ 0.000359) compared to controls (43.5 11.5). This 1.7-fold increase in biomechanical hardness resulted in about 83% of VLRH of native cartilage within 4 weeks. Despite of only a trend to more GAGdeposition, the hardness of LiCl stimulated scaffolds (58.1 7.2; *P ¼ 0.00062) was slightly but significantly elevated compared to controls [Fig. 3(A)]. Overall, a significant correlation between GAGcontent and absolute biomechanical hardness was established in chondrocyte-seeded TE-constructs (Pearson correlation, r ¼ 0.76, *P ¼ 0.000359) [Fig. 3(B)]. FE optimized parameter identification further displayed that BMP-4/-7-stimulated constructs presented a higher mechanical


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Fig. 3. Effect of BMP-4/-7 and LiCl treatment on biomechanical properties of chondrocyte-seeded TE-constructs. After seeding collagen sponges with 1.5 106 porcine chondrocytes, scaffolds were cultured for 4 weeks as indicated in Fig. 2. (A) Evaluation of biomechanical properties by the Digi-Test-II testing device following the VLRH principle. Presentation of absolute hardness values VLRH at the last loading point during measurement (300 ms) (n ¼ 8 per group). ManneWhitney U test, Bonferroni post-hoc. Control vs BMP-4/-7 *P ¼ 0.001; Control vs LiCl *P ¼ 0.016; BMP-4/-7 vs LiCl *P ¼ 0.002. Data are presented as BoxeWhisker plots. (B) Correlation between the absolute GAG-content per scaffold and absolute hardness values. C control, : LiCl, - BMP-4/-7.

compliance, according to a significantly increased poroelastic E modulus (105.73 34.05 kPa; 13.3-fold; *P ¼ 0.000001) compared to controls (7.95 4.17 kPa) [Fig. 4(A)]. Further, significantly enhanced Poisson’s ratio (n) of the BMP-4/-7 treated specimens (0.0703 0.0409; *P ¼ 0.013) compared to controls (0.0432 0.0284) [Fig. 4(B)] indicated that the scaffolds became less compressible under BMP-4/-7 stimulation. Significantly lower hydraulic permeability (k) was detected for BMP-4/-7-stimulated constructs (5.8 2.1056 1014 m^4/N.s; *P ¼ 0.000001) compared to control samples (4.4 3.1289 1013 m^4/N.s) [Fig. 4(C)]. LiCl treatment did not significantly improve biomechanical properties according to poroelastic E modulus (10.46 9.26 kPa), Poisson’s ratio (n) (0.0294 0.0299) or hydraulic permeability (k) (3.3 3.3277*1013 m^4/N.s) (Fig. 4). Discussion Cartilage TE-constructs generated in vitro for implantation into chondral defects should ideally closely imitate the physiologic functional characteristics of native cartilage. Therefore, a number of studies aimed to enhance functional properties of engineered tissue to recreate characteristics of native cartilage in vitro11,46e48. As the unique capacity of cartilage is to uniformly distribute and to absorb mechanical forces affecting the joint, it is of great importance to elucidate biomechanical characteristics of in vitro generated tissue. To mimic functional properties of native cartilage, mechanical consequences of enhanced ECM-deposition caused by diverse stimuli have to be distinguished. Although several potent biological stimulators of chondrocyte metabolism were used to promote ECM-deposition in TE-constructs, their effect on biomechanical properties of engineered cartilage and thus their value for functional cartilage TE to date remain poorly defined. We here analysed whether activation of BMP- or Wnt-signalling is effective to enhance biomechanical properties of chondrocyteseeded collagen matrix-based TE-constructs and found that stimulation of the BMP-signalling pathway by the BMP-4/-7 heterodimer effectively enhanced functional characteristics of in vitro generated tissue. Actually chondrogenic standard culture conditions, effective to deposit an increasing amount of ECM in TE-constructs over time, were insufficient to generate TE-constructs exhibiting biomechanical characteristics of native tissue within 6 weeks. According to extrapolation from a biomechanical standard curve generated in this study, estimated 4 months of culture under standard conditions would be required to approximately reach the hardness of native cartilage. This underlines that, in order to be efficient,

additional stimulation is essential to improve functional TE of cartilage. Probing BMP-signalling and Wnt-stimulation as potent alternatives, BMP-signalling via the BMP-4/-7 heterodimer was very successful to enhance biomechanical properties of chondrocyte-seeded constructs, while the induction of Wntsignalling was less effective. Under continuous use of BMP-4/-7 in chondrogenic medium, 83% of native cartilage hardness was reached within 4 weeks along with a significantly enhanced stiffness and reduced hydraulic permeability compared to standard culture. Since about 9 weeks of culture would be required to reach similar biomechanical characteristics under standard conditions according to the standard curve, BMP-4/-7 stimulation, thus, reduced culture time by more than 50%. These promising results will now encourage further investigation of biological factors for their potency in functional TE of cartilage in order to improve current regeneration strategies in clinical use for treatment of focal cartilage defects and early OA lesions. To our knowledge, the current study is the first to demonstrate that a BMP heterodimer can significantly enhance ECM-deposition in chondrocyte-seeded TE-constructs. BMP-4/-7 was here used at 100 ng/mL, based on past studies where BMP-7 concentrations of 50e100 ng/mL appeared to be optimal in inducing an anabolic response in chondrocytes. Additionally, the concentration of BMP-7 in synovial fluid and cartilage samples of human adults was found to be within this range49,50, suggesting that 100 ng/mL displays a physiological dose. For BMP-4 stimulation, 30e100 ng/mL were applied to affect ECM-production by chondrocytes20,51. Aiming to analyse biomechanical consequences of factor stimulation, we decided to work with a rather high BMP heterodimer concentration in order to induce a profound effect translating into biomechanical differences. Since the literature already established the superiority of BMP-4/-7 heterodimers above homodimers, we refrained from a systematic analysis of the exact advantage BMP-4/-7 heterodimers may bring above BMP-4 and BMP-7 homodimers. Respective data for cartilage will, however, be interesting and should be evaluated in dose response studies to reduce costs for ECM stimulation in chondrocyte-seeded TE-constructs in the future. Although the morphological organisation of native cartilage may hardly be exactly imitated in TE-constructs, the orientation of matrix embedded chondrocytes into parallel vertical columns was here supported by the chosen collagen type I/III carrier which introduced parallel collagen layers as guiding tracks for cell seeding. Whether this optimized cell orientation may have further contributed to the promising biomechanical characteristics of our constructs remains to be determined by comparison with randomly oriented collagen sponges to be tested in future studies.


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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 Author’s contributions statement Q2 104 105 106 Anika Krase: 107 108 (1) Conception and design, Collection and assembly of data, 109 Analysis and interpretation of the data 110 (2) Drafting the article 111 (3) Final approval of the article 112 113 Reza Abedian: 114 115 (1) Collection and assembly of data, Analysis and interpretation 116 of the data 117 (2) Critical revision of the article for important intellectual 118 content 119 (3) Final approval of the article 120 121 Eric Steck: 122 123 (1) Conception and design 124 (2) Critical revision of the article for important intellectual 125 content 126 (3) Final approval of the article 127 128 Christof Hurschler: 129 130 (1) Conception and design method to examine functional quality characteristics of cartilage TE-constructs. Molar concentrations between 5 mM and 10 mM LiCl have been shown to be effective in inducing the canonical Wntsignalling pathway30,31,52, but testing of only one high LiCl dosage is a limitation of this study. Alternative molecules activating Wntsignalling in chondrocytes may be considered in the future. Because biomechanical parameters are basically a consequence of ECM-deposition7, and proteoglycans are of main importance for tissue swelling-pressure2, the total GAG-amount was here used as a correlate for ECM-deposition. As collagen type II is the pivotal second component of cartilage, raising the total collagen type II content by BMP-4/-7 stimulation of COL2A1 gene expression may further have contributed to increased absolute hardness values. BMP and Wnt-activation are known to stimulate both proteoglycan and collagen type II production15,19,31,46,52. In view of the presence of an excess of collagen type I and III from the carrier we, however, refrained from quantifying this second most important biochemical ECM component on the protein level. In summary, BMP-4/-7 heterodimer stimulation of chondrocyteseeded scaffolds represents a promising strategy to improve functional TE of cartilage. Our interdisciplinary work is unique in combining a collagen scaffold with parallel fibre orientation to support chondrocyte column formation with signalling via a so far untested BMP heterodimer and Wnt agonist with novel and innovative testing of biomechanical parameters of engineered tissue. Resulting TE-constructs displayed significantly enhanced biochemical and functional biomechanical properties approximating the values of native cartilage already after few weeks of culture reducing culture time by more than 50% compared to standard conditions. Current studies are now under way to optimize this application under use of clinically more relevant human chondrocytes. Preliminary data demonstrate that BMP-4/-7 heterodimer is also effective on human chondrocytes and when applied locally. Therefore, future testing should reveal whether BMP-4/-7 loaded carriers might offer the possibility for an earlier transplantation of less mature constructs into chondral defects of patients rather than mature functional cartilage tissue from in vitro culture.

Fig. 4. Biomechanical properties of BMP-4/-7 and LiCl-treated cartilage TE-constructs. After chondrogenic culture and stimulation either with BMP-4/-7 or LiCl until day-42, constructs were evaluated by confined compression analysis. Stress relaxation testing was performed using in a displacement rate of 0.15 mm/s to a displacement step of 0.5 mm resulting in 25e30% of axial strain, and held at this deformation until equilibrium stress was attained. A total of 55 scaffolds seeded with cells of two donors were measured yielding 55 confined compression data points. A custom FE-based optimized parameter extraction routine was applied to derive the material parameters of a biphasic poroelastic material model. ManneWhitney U test, Bonferroni post-hoc. Data are presented as BoxeWhisker plots. (A) Calculation of the poroelastic E Modulus provides information about construct stiffness (Control vs BMP-4/-7 *P ¼ 0.000001; BMP-4/-7 vs LiCl *P ¼ 0.000001), (B) the Poisson’s ratio about compressibility (Control vs BMP-4/-7 *P ¼ 0.013; BMP-4/-7 vs LiCl *P ¼ 0.015), (C) and the hydraulic permeability about the ability of water to flow under pressure (Control vs BMP-4/-7 *P ¼ 0.000001; BMP-4/-7 vs LiCl *P ¼ 0.000014).

In contrast to the strong stimulating influence of BMP-4/-7 heterodimer, biochemical and biomechanical consequences of Wnt-activation in chondrocyte-seeded collagen matrices were disappointing. Significantly enhanced-content of LiCl-treated vs control scaffolds (1.3-fold, *P ¼ 0.003) and up-regulation of Wntresponsive AXIN2 gene expression demonstrated that the chosen LiCl concentration was not toxic but effective. It resulted merely in a trend towards higher GAG-deposition which, however, seemed sufficient to slightly but significantly enhance absolute hardness values of the constructs. This demonstrated that the here introduced indentation device Digi-Test-II is a precise and sensitive


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(2) Critical revision of the article for important intellectual content, Obtaining of funding (3) Final approval of the article

12.

Wiltrud Richter: 13. (1) Conception and design, Analysis and interpretation of the data (2) Critical revision of the article for important intellectual content, Obtaining of funding, Drafting the article (3) Final approval of the article

14.

Following authors take responsibility for the integrity of the work as a whole, from inception to finished article: Anika Krase: [email protected] Prof. Dr Wiltrud Richter: [email protected]

15.

Conflict of interest Authors disclose any commercial or other potential conflict of interest regarded to this work.

16.

17. Acknowledgements The authors thank Jessica Gabler for excellent technical assistance and Simone Gantz for professional statistical support. This work was supported by the German Ministry of Education and Research (BMBF grant 0315579 and BMBF grant 0315577H), and the Orthopaedic University Hospital Heidelberg.

18.

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