bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 PTH decreases in vitro human cartilage regeneration without affecting hypertrophic 2 differentiation 3 4 Marijn Rutgers1, Frances Bach2, Luciënne Vonk1, Mattie van Rijen1, Vanessa Akrum1, 5 Antonette van Boxtel1, Wouter Dhert1,2, Laura Creemers1 6 7 1 Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands 8 2 Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, 9 Utrecht University, Utrecht, the Netherlands 10 11 Address for correspondence 12 Dr. F.C.Bach 13 Utrecht University, Faculty of Veterinary Medicine, 14 Yalelaan 104, 3584 CM Utrecht, the Netherlands 15 [email protected], +31302537563 16 1 bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract 19 Regenerated cartilage formed after Autologous Chondrocyte Implantation may be of 20 suboptimal quality due to postulated hypertrophic changes. Parathyroid hormone-related 21 peptide, containing the parathyroid hormone sequence (PTHrP 1-34), enhances cartilage 22 growth during development and inhibits hypertrophic differentiation of mesenchymal stromal 23 cells (MSCs) and growth plate chondrocytes. This study aims to determine whether human 24 articular chondrocytes respond correspondingly. Healthy human articular cartilage-derived 25 chondrocytes (n=6 donors) were cultured on type II collagen-coated transwells with/without 26 0.1 or 1.0 μM PTH from day 0, 9, or 21 until the end of culture (day 28). Extracellular matrix 27 production, (pre)hypertrophy and PTH signaling were assessed by RT-qPCR and/or 28 immunohistochemistry for collagen type I, II, X, RUNX2, MMP13, PTHR1 and IHH and by 29 determining glycosaminoglycan production and DNA content. The Bern score assessed 30 cartilage quality by histology. Regardless of the concentration and initiation of 31 supplementation, PTH treatment significantly decreased DNA and glycosaminoglycan 32 content and reduced the Bern score compared with controls. Type I collagen deposition was 33 increased, whereas PTHR1 expression and type II collagen deposition were decreased by 34 PTH supplementation. Expression of the (pre)hypertrophic markers MMP13, RUNX2, IHH 35 and type X collagen were not affected by PTH. In conclusion, PTH supplementation to 36 healthy human articular chondrocytes did not affect hypertrophic differentiation, but 37 negatively influenced cartilage quality, the tissues’ extracellular matrix and cell content. 38 Although PTH may be an effective inhibitor of hypertrophic differentiation in MSC-based 39 cartilage repair, care may be warranted in applying accessory PTH treatment due to its effects 40 on articular chondrocytes. 41 42 2 bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 43 Introduction 44 Autologous chondrocyte implantation (ACI) is an effective treatment in patients with 45 medium-sized cartilage defects (1). Chondrocytes isolated from healthy non weight-bearing 46 cartilage and re-transplanted after in vitro expansion ideally fill the void with hyaline 47 neocartilage (2). Variable results have, however, been found regarding the obtained cartilage 48 quality, with fibrous or even hypertrophically differentiated tissue instead of healthy cartilage 49 (3). Similarly, MSC-based regeneration either as part of microfracture procedures or as 50 exogenous cell source has been shown to result in hypertrophic differentiation (4). A possible 51 tool to prevent this unfavorable differentiation pathway may be the co-administration of 52 parathyroid hormone related-peptide (PTHrP). 53 PTHrP plays an important role in early development and limb growth (5) and is crucial in 54 maintaining the chondrocytic phenotype in native cartilage. In the growth plate, a highly 55 organized cartilage structure that enables longitudinal bone growth, PTHrP maintains 56 chondrocytes in a proliferating state and prevents hypertrophic differentiation and bone 57 formation (6). Parathyroid hormone (PTH) is assumed to have similar effects as PTHrP (7,8), 58 as they share their N-terminus and receptor (PTHR1) (9,10). Both PTH and PTHrP can 59 enhance cartilage formation by stimulating the expression of SRY-box 9 (6) (SOX9, 60 transcription factor required for chondrocyte differentiation and cartilage formation (11)) and 61 by increasing cell proliferation through induction of cyclin D1 (CCND1) (12). PTHrP/PTH 62 have been demonstrated to stimulate chondrogenic differentiation of mesenchymal stromal 63 cells (MSCs) and to prevent hypertrophic differentiation of MSCs (13-17) and growth plate 64 chondrocytes (18,19) in vitro. Lastly, in rabbit osteochondral defects, intra-articular PTH 65 administration stimulated tissue regeneration in vivo (20,21). In contrast to osteochondral 66 defect healing, ACI is based on chondrocyte implantation, either or not supplemented with 67 MSCs (22). Although expanding chondrocytes have stem cell-like properties (23), 3 bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 68 multilineage (especially osteogenic and adipogenic) differentiation efficacy is very low 69 compared with MSCs, which may indicate that a chondroid precursor, but not a multipotent 70 mesenchymal cell type is present in expanding chondrocytes (24). 71 As yet, it is unknown whether PTH may have similar effects on articular chondrocyte- 72 mediated regeneration in terms of inhibition of hypertrophy and stimulation of regeneration. 73 Therefore, in order to define whether PTH holds promise as an additive treatment strategy to 74 current challenges faced by ACI, this study determined the effects of PTH on expanded 75 human articular chondrocytes in an in vitro model of cartilage regeneration. 76 77 Materials and Methods 78 Human chondrocytes 79 Healthy human femoral knee cartilage of three male and three female donors (mean age 68, 80 range 47-83 years) was obtained post-mortem. Only macroscopically intact (Collins grade 0-1 81 (25)) cartilage was used (four samples grade 0, two samples grade 1). Collection of all patient 82 material was done according to the Medical Ethical regulations of the University Medical 83 Center Utrecht and according to the guideline ‘good use of redundant tissue for clinical 84 research’ constructed by the Dutch Federation of Medical Research Societies on collection of 85 redundant tissue for research (www.fedara.org). This study does not meet the definition of 86 human subjects research or require informed consent. Anonymous use of redundant tissue for 87 research purposes is part of the standard treatment agreement with patients in our hospital 88 (26). 89 90 Chondrocyte isolation and expansion 91 Cartilage was digested overnight in Dulbecco’s modified Eagle Medium (DMEM; 42430, 92 Invitrogen) containing 0.1% w/v collagenase type II (CLS2, Worthington), 1% v/v 4 bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 penicillin/streptomycin (P/S; 15140, Invitrogen). Isolated chondrocytes were washed in PBS 94 and expanded at 5000 cells/cm2 in DMEM containing 1% P/S, 10% v/v fetal bovine serum 95 (DE14-801F, Lonza), 10 ng/mL FGF (223-FB, R&D Systems). At 80% confluency, cells 96 were trypsinized and passaged. Passage two cells were used for redifferentiation culture or 97 snap-frozen for RNA analysis. 98 99 Redifferentiation culture 100 Since more recent ACI versions are based on collagen carriers (27), the chondrocytes were 101 cultured on collagen type II-coated membranes in a 24-wells transwell system as described 102 previously (28). Passage two chondrocytes were seeded on inserts (PICM01250, Millipore) 103 with a hydrophilic poly-tetrafluoroethylene (PTFE) membrane at 1.6 x106 cells/cm2 in 104 DMEM with 2% w/v ITSx (51500, Invitrogen), 2% w/v ascorbic acid (A8960, Sigma- 105 Aldrich), 2% w/v human serum albumin (HS-440, Seracare Life Sciences), 100 units/mL 106 penicillin, 100 μg/mL streptomycin, and 10 ng/mL TGF-2 (302-B2, R&D Systems). Before 107 culture, the membranes had been coated with 0.125 mg/mL type II collagen (C9301, Sigma- 108 Aldrich) in 0.1 M acetic acid. After thorough rinsing, efficient coating was verified by 109 immunohistochemistry (29). To mimic the different phases of maturation, PTH was supplied 110 at different time points in two different concentrations: 0.1 or 1.0 μM PTH (based on 111 Kafienah et al. (2007) (13)) was added every media change (three times a week) from days 0 112 (n=4), 9 (n=6) or 21 (n=6) onwards, whereas the controls (n=6) did not receive PTH. After 28 113 days, the tissues were fixed in 10% w/v neutral buffered formalin (for histological analysis) 114 or snap-frozen and stored at -20 ºC (glycosaminoglycan (GAG) and DNA content analysis) or 115 -80 ºC (for RNA analysis). 116 117 Gene expression analysis 5 bioRxiv preprint doi: https://doi.org/10.1101/560771; this version posted February 26, 2019.