Despite possessing resident progenitors committed to chondrogenesis, articular cartilage has poor intrinsic regenerative capability and minor cell turnover, mainly due to its avascular and alymphatic nature [1
]. The isolation from the systemic circulation keeps away the inflammatory and reparative mechanisms triggered after injury, precluding damaged cartilage from recovering its functional configuration, which leads to tissue degeneration and the subsequent establishment of osteoarthritis (OA). Nowadays, OA is one of the major musculoskeletal diseases [2
] and affects joints by causing gradual loss of articular cartilage together with osteophyte growth and synovial inflammation [3
]. Since articular cartilage works as lubricant and load-bearing surface in healthy joints, symptoms of these disorders are pain and disability, which lead to an impaired patients’ quality of life and increased health-care costs for the society.
Currently, several therapeutic approaches for focal chondral defects and OA are used, including bone marrow surgical stimulation aimed at triggering intrinsic reparative mechanisms, cell therapy based on autologous chondrocyte implantation (ACI) and osteoarticular auto/allografts to fill and restore cartilage defects [4
]. However, the treatment of extended defects is still challenging, especially for elderly people, and these techniques are still defective because the neo-tissue is often fibrotic and not completely integrated with neighboring tissues [5
Therefore, research efforts are directed to explore new methods to achieve articular cartilage regeneration and repair. The use of therapies based on stem cells has attracted great interest since it is widely known that mesenchymal stem cells (MSCs), such as bone-marrow-derived MSCs (BM-MSCs), can differentiate into the chondrogenic lineage both in vitro and in vivo [6
]. However, stem cell niches are located in situ, where they could participate directly in tissue homeostasis and repair processes. In fact, joint tissues, such as the synovium, hold MSCs with chondrogenic potential [8
], and articular cartilage itself contains a postnatal progenitor cell population [9
]. Moreover, during OA, pathological joint tissues and synovial fluid become enriched of cells with features of stemness [11
], probably as an attempt to limit the ongoing damage and restore tissue integrity.
In the field of regenerative medicine, local transplantation or systemic infusion of stem cells represents an effective cellular therapy in many pathological states concerning the musculoskeletal system. Nevertheless, regeneration strategies targeting stem cells in situ could be more attractive and more advantageous thanks to the absence of in vitro culture steps and loss of injected cells. According to this approach, endogenous stem cells are recruited to the injury site by administration of bioactive factors. Thus, in the last decades, among a wide range of products, platelet-rich plasma (PRP) has spread as a clinical treatment tool for musculoskeletal diseases [13
]. Since PRP or other platelet derivatives (i.e., platelet lysate (PL)) are a mix of growth factors, cytokines and chemokines normally involved in tissue healing, the rationale behind their application is the reactivation of latent endogenous regenerative mechanisms. Several studies have investigated PRP or PL roles both in vitro and in vivo, highlighting their capacity to exert anti-inflammatory and proliferating effects on cells [14
], as well as to stimulate resident progenitors or to recruit circulating ones (together with immune and endothelial cells) [17
Regarding cartilage disorders, treatments based on platelet-derived products have shown pain relief and functional improvement in patients, confirming their chondroprotective function in these pathologies [19
]. From a mechanistic point of view, these beneficial outcomes could be explained by the fact that PRP-derived factors promote matrix deposition and downregulate inflammatory signalling in chondrocytes [22
], and enhance migration and chondrogenic differentiation of progenitor cells [17
] as well as cartilage tissue maturation [24
However, the mechanism of action and efficacy of platelet derivatives in orthopaedics still need to be elucidated, especially due to the wide variety of preparation and standardization methods that can impact product composition [25
], thereby affecting the physiological response. A better understanding of events leading to PRP- or PL-induced cartilage repair may allow solving these issues.
Here, we report that ex vivo treatment of human articular cartilage from hip joints with PL induces activation and outgrowth of cells that are endowed with some features of stemness, such as clonogenicity and expression of nestin [26
], and higher proliferation capacity than resident chondrocytes with concurrent chondrogenic potential maintenance, both in vitro and in vivo. Stimulation of nestin-positive progenitor cells induced by PL in articular cartilage is of special interest for the future development of therapeutic strategies given the involvement of these cells in tissue regenerative processes. Moreover, we further characterize the PL effects on the phenotype of mature hip articular chondrocytes by showing that they reverted to an earlier stage similar to that of chondro-progenitor cells.
2. Materials and Methods
2.1. Platelet Lysate (PL) Preparation
Buffy coat samples obtained from the whole blood of healthy donors at the Blood Transfusion Center of the IRCCS Policlinico San Martino Hospital (Genova, Italy) were used to prepare PL. All the procedures were performed with the approval of the Institutional Ethics Committee and the Italian Ministry of Health: no. 423/2017-PR -7/7/2016, (D. lgs. 26/2014). According to Backly et al. [27
], platelet pellet was obtained after serial centrifugation and resuspended at a concentration of 1 × 107
platelets/µL in plasma to get PRP. Platelet membrane rupture in the PRP suspension was achieved by three cycles of immersion in liquid nitrogen for 1 min and incubation at 37 °C for 6 min. The suspension was centrifuged at 900 × g
for 3 min at 4 °C and the supernatant was collected to obtain the PL, divided in aliquots and stored at −20 °C until use. Further details on platelet product standardization and safety were reported in [28
In preliminary studies, several PL concentrations were tested (from 2.5 to 10%) on chondrocyte and cartilage cultures (data not shown). Five percent PL represents the maximum effective concentration in terms of cell responses (proliferation and outgrowth from tissue chips).
2.2. Cell Primary Cultures
2.2.1. Chondro-Progenitor Cells (CPCs)
Human articular cartilage biopsies were harvested from patients (N
= 20 with an age range from 31 to 88 years old, 65-year median age) undergoing hip replacement surgery. All tissue samples were obtained with written informed patients’ consent and according to the guidelines of the institutional Ethics Committee of IRCCS Policlinico San Martino Hospital (Genova, Italy), no. 423/2017-PR -7/7/2016. Articular cartilage was separated from subchondral bone and fragmented in slices, which were further cut into disks with a biopsy punch of 8 mm in diameter. Each disk was divided into two halves, and each half was then cultured in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM HG) containing 1 mM sodium pyruvate, 100 mM HEPES buffer, 1% penicillin/streptomycin and 1% L-glutamine (all from Euroclone, Milano, Italy) supplemented either with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) or 5% PL in 6-well plates for 1 month (Figure 1
A). Putative chondro-progenitor cells (CPCs), moving from cultured cartilage chip to the dish, were detached with trypsin/EDTA (Euroclone, Milano, Italy) and expanded in aforementioned medium supplemented with 5% PL (CPCs-PL).
2.2.2. Primary Articular Chondrocytes (ACs)
Primary articular chondrocytes (ACs) were obtained as described by Pereira et al. [14
] from the remaining cartilage biopsy (Figure 1
A). In brief, chondrocytes were released by repeated digestions using an enzymatic solution composed of 1 mg/mL hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), 400 U/mL collagenase I, 1000 U/mL collagenase II (both from Worthington Biochemical, Lakewood, NJ, USA) and 0.25% trypsin (Thermo Fisher Scientific, Waltham, MA, USA). The cells obtained were plated in DMEM HG basal medium described above and containing 10% FBS. At ~90% of confluence, cells were trypsinized and split in culture medium supplemented with 10% FBS (ACs-FBS) or 5% PL (ACs-PL). During culture, cells were monitored using a bright-field microscope equipped with a digital camera (Leica DMi1; Leica Microsystems, Wetzlar, Germany). All described cell cultures were maintained in an incubator at 37 °C, with 5% CO2
and in normoxic oxygen condition.
2.3. Growth Kinetics
Growth kinetics were determined by plotting cell doublings of ACs-FBS, ACs-PL and CPCs-PL against time. Cell doublings were calculated considering the number of cells plated and recovered at each passage. Briefly, semi-confluent cells were trypsinized, counted and always replated at a density of 1.25 × 104 cells/cm2 in 60 mm culture dishes. Six primary cultures were tested (N = 6).
2.4. Western Blot Analysis
At passage 2, confluent monolayers of ACs-FBS, ACs-PL and CPCs-PL were washed with phosphate-buffered saline 1X (PBS) and scraped in cold radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris (pH 7.5), 150 mM sodium chloride, 1% deoxycholic acid, 1% triton X-100, 0.1% SDS, 0.2% sodium azide and proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein extract concentration was quantified by Bradford assay (Serva Electrophoresis GmbH, Heidelberg, Germany) and Western blot was performed according to Nguyen et al. [30
]. Equal amounts of total proteins (10 µg) were loaded on 4–12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA), and electrophoresis was performed. Gels were blotted onto nitrocellulose membranes (GE Healthcare Life Sciences, Uppsala, Sweden), immunoprobed overnight at 4 °C with primary antibodies raised against cyclin D1 (Abcam, Cambridge, UK) and α-tubulin (Sigma-Aldrich, St. Louis, MO, USA), both at a 1:10,000 dilution. After washing, membranes were exposed to horseradish peroxidase-linked goat anti-rabbit IgG at dilution of 1:5000 (GE Healthcare Life Sciences, Uppsala, Sweden) for 1 h at room temperature (RT), and bands were visualized using enhanced chemiluminescence (ECL, GE Healthcare Life Sciences, Uppsala, Sweden). Then, X-ray films (Fujifilm GmbH, Düsseldorf, Germany) were exposed to membranes, developed and fixed. Three primary cultures were tested (N
2.5. Evaluation of Cell Senescence
ACs-FBS, ACs-PL and CPCs-PL were analysed for senescence by detection of the senescence-associated β-galactosidase (SA-βgal) activity in a chromogenic assay (Sigma-Aldrich, St. Louis, MO, USA) according to previous protocol [31
]. In brief, adherent cells in 24-well dishes were fixed in 3% paraformaldehyde (PFA) and stained overnight at 37 °C with fresh staining solution containing 40 mM citric acid/sodium phosphate buffer, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, 2 mM magnesium chloride and 1 mg/mL 5-bromo-4-chloro-3-indolyl-d
-galactoside (X-gal) in distilled water (all reagents from Sigma-Aldrich, St. Louis, MO, USA). Positive cells were observed under microscope Axiovert 200M (Carl Zeiss, Oberkochen, Germany) and counted in five regions of interest (ROI) at 20× magnification for each replicate. Stained cells were calculated as a percentage of the total number of cells on the plate. Cells from 5 donors were subjected to the assay at passage 2 (N
2.6. Assay for In Vitro and In Vivo Neoplastic Transformation of CPCs
To exclude malignant properties of CPCs, in vitro colony assay formation and in vivo tumorigenesis were investigated. For in vitro test, anchorage-independent growth assay in methylcellulose media was conducted [32
]. Thus, CPCs-PL at passage 3 were plated at a density of 10,000 cells/35 mm petri dishes in the semi-solid culture system provided by StemMACS HSC-CFU Media (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. After 14 days of incubation, formation of colony-forming unit (CFU)-cells (CFU-C) was assessed under an inverted microscope (Leica DMi1; Leica Microsystems, Wetzlar, Germany). Three primary cultures were tested (N
= 3). MDA-MB-231 triple-negative breast cancer cell line was used as positive control. In vivo tumorigenesis was assessed with CPCs-PL at passage 2 from two different pools. For each pool, 1 × 106
cells were injected subcutaneously into NOD/SCID mice (N
= 12 animals, males and females, 6–8 weeks old). The mice were monitored up to 2–3 months. All experimental animal procedures were evaluated and approved by Ethics Committee for animal experimentation (CSEA) and communicated to the Italian Ministry of Health in accordance with article 31 of the D. lgs 26/2014 (approval no. 29/2017-PR, 16/01/2017 by Italian Ministry of Health).
2.7. RNA Extraction and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA from ACs-FBS, ACs-PL and CPCs-PL at passage 1 grown upon confluence (in 100 mm dishes) was extracted by TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. RNA concentrations were measured at 260 nm using Nanodrop TM 1000 (Thermo Fisher Scientific, Waltham, MA, USA) and RNA purity was checked considering 260 nm/280 nm ratio with values included in the 1.5–2.1 range. Complementary DNA (cDNA) synthesis was performed starting from 1 μg of total RNA and using SuperScript First-Strand synthesis system for reverse transcription polymerase chain reaction (RT-PCR) (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instruction. Transcript levels of target genes were measured by real-time quantitative PCR (qRT-PCR) using Power SYBR®
Green PCR Master Mix on 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The housekeeping gene GAPDH
, commonly utilized as a reference gene in cartilage and bone qPCR assays [33
], was used as the endogenous control for normalization. The selected human-specific primer sequences were as follows: type II collagen (COL2A1
), forward 5′ - GGCAATAGCAGGTTCACGTACA - 3′, reverse 5′ - CGATAACAGTCTTGCCCCACTT - 3′; type I collagen (COL1A1
), forward 5′ - CAGCCGCTTCACCTACAGC - 3′, reverse 5′ - TTTTGTATTCAATCACTGTCTTGCC - 3′; SOX9
, forward 5′ - CCCGCACTTGCACAACG - 3′; reverse 5′ - TCCACGAAGGGCCGCT - 3′; nestin (NES
), forward 5′ - CAGAGGTGGGAAGATACGGT - 3′, reverse 5′ - AGCTCTGCCTCATCCTCATT - 3′; GAPDH
, forward 5′ - CCATCTTCCAGGAGCGAGAT - 3′, reverse 5′ - CTGCTTCACCACCTTCTTGAT - 3′. Data were reported as ratio GENE
, which represents the expression of the gene of interest divided by the expression of GAPDH
in the same sample.
2.8. Immunofluorescence Staining and Immunophenotypic Characterization by Flow Cytometry
To perform immunofluorescence staining, ACs-FBS, ACs-PL and CPCs-PL at passage 1 were seeded on coverslips at a density of 105 cells/cm2 and fixed with 3.7% PFA after 3–4 days of culture. Fixed cells were permeabilized with a solution containing 20 mM HEPES (pH 7.4), 300 mM sucrose, 50 mM sodium chloride, 3 mM magnesium chloride and 0.5% triton X-100. After blocking with 20% normal goat serum (NGS, Thermo Fisher Scientific, Waltham, MA, USA), samples were incubated overnight at 4 °C with primary antibodies raised against SOX9, 1:200 diluted in 10% NGS (Abcam, Cambridge, UK); type II collagen, 1:250 diluted in 10% NGS (CIICI-Developmental Studies Hybridoma Bank, University of Iowa); type I collagen, 1:300 diluted in 10% NGS (SP1.D8-Developmental Studies Hybridoma Bank, University of Iowa); nestin, 1:2000 diluted in 10% NGS (Abcam, Cambridge, UK); and cleaved caspase-3, 1:400 diluted in 10% NGS (Cell Signaling Technology, Danvers, MA, USA). Positive staining was detected by incubation with Alexa Fluor 488- or Alexa Fluor 594-conjugated anti-mouse or anti-rabbit immunoglobulin IgG secondary antibodies diluted 1:300 in NGS 10% (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT, followed by nuclear labelling with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Negative control staining was performed without primary antibody incubation. Samples were observed under epifluorescent illumination using an Axiovert 200M microscope, and images were captured with AxiocamHR camera (Carl Zeiss, Oberkochen, Germany).
Expanded ACs-FBS, ACs-PL and CPCs-PL at passage 2 were phenotypically characterized for a set of surface markers using flow cytometry. After trypsinization, 1 × 105 cells were incubated separately with 1 µL of one of the following fluorescein isothiocyanate (FITC)- or phycoerythrine (PE)-conjugated antibodies: CD44-FITC, CD166-PE, HLA-ABC-PE, HLA-DR-FITC (all from BD Pharmigen), CD90-PE, CD105-PE, CD73-FITC, CD146-FITC, CD106-PE, CD45-FITC, CD34-PE, CD29-PE and isotype-matched IgG-PE and IgG-FITC control antibodies (all from eBiosciences/ ThermoFisher Scientific, Waltham, MA, USA). The staining was performed for 30 min at 4 °C in the dark to preserve the fluorochromes. Samples were run on a CyAN ADP cytofluorimeter (Beckman-Coulter, Brea, CA, USA). Data were analyzed using FlowJo V10 software (Tree Star Inc., San Carlos, CA, USA) and expressed as Log fluorescence intensity versus number of cells. Experiments were repeated on three different primary cultures (N = 3).
2.9. Colony-Forming Unit Fibroblast (CFU-F) Assay
Clonogenic potential of ACs-FBS, ACs-PL and CPCs-PL at passage 1 was explored by plating them at low density (10 cells/cm2) in 100 mm culture dishes and performing the colony staining after 12 days of culture. At the end of the culture time, cells were washed with PBS, fixed with 3.7% PFA in PBS for 15 min at RT and stained with 1% methylene blue in borate buffer (10 nM, pH 8.8) for 45 min at RT. CFU-F assay was performed in duplicate for each tested primary culture (N = 10). Colony-forming efficiency (CFE%) was calculated as follows: (number of colonies formed/number of plated cells) × 100. A set of 6-well dishes were prepared for detection of nestin by immunofluorescence.
2.10. In Vitro Multilineage Differentiation Potential
The chondrogenic potential of CPCs grown in 5% PL and ACs expanded in the presence of either FBS or PL was checked at passage 1 by micromass pellet culture in vitro (N
= 3). About 2.5 × 105
cells were pelleted in conical tubes and cultured for 3 weeks in chondrogenic medium containing 10 ng/mL human transforming growth factor-β1 (hTGF-β1) (PeproTech, Rocky Hill, NJ, USA), 10−7
mol/L dexamethasone and 50 mg/mL ascorbic acid (both from Sigma-Aldrich, St. Louis, MO, USA) according to Johnstone et al. [6
]. Chondrogenic differentiation was subsequently investigated by histological staining with toluidine blue (see Section 2.12
To test osteogenic differentiation, cells were seeded in 24-well plates at a density of 105 cells/cm2 in the presence of osteogenic induction medium containing 5 μg/mL ascorbic acid, 10−7 mol/L dexamethasone and 10 mmol/L β-glycerophosphate (all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of culture, calcium deposits were stained by Alizarin Red S (Sigma-Aldrich, St. Louis, MO, USA) solution.
To induce adipogenesis, cells were seeded as just reported above and grown in culture medium containing 1 μmol/L dexamethasone, 60 μmol/L indomethacin, 10 μg/mL insulin and 1 mmol/L 3-Isobutyl-1-methylxanthine (IBMX) (all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of culture, intracellular lipid drops were detected with Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA). Osteogeninc and adipogenic potential of CPCs and ACs were determined at passage 2 in triplicate on three different primary cell cultures (N = 3).
2.11. In Vivo Cartilage and Bone Formation
CPCs-PL chondrogenic and osteogenic potential in vivo was investigated by implantation of cell pellets and cell-seeded biomaterials in athymic mice (female CD-1 nu/nu, 6–8 weeks old; Charles River Laboratories Italia, Lecco, Italy). CPC pellets were obtained as already described in Section 2.10
and implanted subcutaneously in mice after three days of in vitro culture in chondrogenic medium.
Moreover, CPCs-PL at passage 2 were also seeded on absorbable polyglycolic-acid–hyaluronan (PGA-HA) scaffolds (BioTissue AG, Zurich, Switzerland) to detect cartilage formation and/or on calcium phosphate ceramic scaffolds (hydroxyapatite/β-tricalcium phosphate, HA/β-TCP, Biomatlante, Vigneux de Bretagne, France) for osteogenic induction. Briefly, CPCs-PL were trypsinized at passage 1 and 2 × 106 cells were resuspended in 33% v/v fibrinogen in PBS (Tissucol, Baxter Healthcare, Deerfield, IL, USA). Both types of scaffolds were soaked with CPCs suspension, and fibrinogen was polymerized by the addition of 1:10 v/v thrombin in PBS (Tissucol, Baxter, Healthcare, Deerfield, IL, USA). Constructs with ACs-FBS at passage 1 associated with ceramic granules were also prepared as control. Cell grafts were maintained in chondrogenic or osteogenic medium for 3 days before subcutaneous implantation in mice. A number of at least three primary cultures was used for these experiments. Groups of 8 animals were sacrificed 4 and 8 weeks after surgery for chondrogenesis or osteogenesis, respectively, and the harvested implants were processed for the histological analysis to evaluate cartilage and bone formation. All experimental animal procedures were evaluated and approved by Ethics Committee for animal experimentation (CSEA) and communicated to the Italian Ministry of Health in accordance with article 31 of the D. lgs 26/2014 (approval no. 29/2017-PR, 16/01/2017 by Italian Ministry of Health).
2.12. Histology and Immunohistochemistry
Cartilage chips, pellet and implant samples were fixed in 3.7% PFA in PBS, dehydrated in ethanol, and paraffin-embedded. Cross sections of 5 µm were cut (by using microtome RM2165, Leica Microsystems, Wetzlar, Germany), dewaxed and stained according to the appropriate histological analysis: haematoxylin and eosin staining to observe cell organization and toluidine blue staining to detect sulphated glycosaminoglycans in cartilage. For immunohistochemical analysis, dewaxed sections were treated with methanol/hydrogen peroxide (49:1) solution for 30 min to inhibit endogenous peroxidase activity, then permeabilized with 0.3% triton X-100 in PBS for 10 min and finally incubated with hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 mg/mL in PBS (pH 6.0) for 30 min at 37 °C. After washes in PBS and incubation with 20% NGS for 1 h to inhibit nonspecific binding, the slices were incubated overnight at 4 °C with primary antibodies raised against: type II collagen, 1:250 diluted in 10% NGS (CIICI-Developmental Studies Hybridoma Bank, University of Iowa); type X collagen, 1:1000 diluted in 10% NGS (Abcam, Cambridge, UK); and proliferating cell nuclear antigen (PCNA), 1:200 diluted in 10% NGS (Abnova, Taipei City, Taiwan). The immunobinding was detected by incubation with biotinylated secondary anti-mouse or anti-rabbit antibodies (Dako, Agilent Technologies Inc., Carpinteria, CA, USA) for 30 min at RT followed by treatment with streptavidin-peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PE, USA). Peroxidase activity was finally visualized by 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO, USA) chromogen substrate. Images were acquired by a microscope Axiovert 200M (Carl Zeiss, Oberkochen, Germany) at different magnifications. Data for PCNA quantification were summarized in a histogram reporting the PCNA positive cell percentage per field view for cartilage chips cultivated in 10% FBS or 5% PL (N = 3).
2.13. Production of Cell-Conditioned Media
ACs-FBS, ACs-PL and CPCs-PL at passage 1 were grown until 80% of confluence, extensively washed with PBS and incubated with DMEM HG culture medium without any supplements for 24 h of conditioning. Conditioned media (CM) from each condition (ACs 10% FBS-CM, ACs 5% PL-CM and CPCs 5% PL-CM) were collected and centrifuged at 300 × g for 10 min and then at 2000 × g for 20 min, and supernatants were stored in aliquots at −80 °C. In cytokine array experiment, supernatants in each condition were further concentrated by using Amicon™ Ultra Centrifugal Filter Units with 3KDa molecular weight cut-off (Merck Millipore, Burlington, MA, USA). Amount of proteins in CM from CPCs and ACs was quantified by performing Bradford assay (Serva Electrophoresis GmbH, Heidelberg, Germany).
2.14. Cytokine Identification in Cell Secretome
The release of cytokines and chemokines in CPCs- and ACs-CM was analysed using the Human XL Proteome Profiler™ Array (R&D Systems, Minneapolis, MN, USA) according to the user’s manual. Briefly, membranes spotted with antibodies were incubated with the same amounts (50 µg/mL) of each CM overnight at 4 °C. The following day, detection antibody cocktail was added for 1 h at RT, before visualization using ECL. Quantitative analysis was performed on scanned (Epson perfection 1260 scanner, Seiko Epson Corporation, Nagano, Japan) X-ray films (Fujifilm GmbH, Düsseldorf, Germany) using the Protein Array Analyser plugin available for ImageJ software (US National Institutes of Health, Bethesda, MD, USA). For each membrane, average spot signal density was determined by densitometry, followed by background subtraction and normalization to the reference spots. For a correspondence between a specific molecule and its position in the membrane, a reference to the manufacturer’s datasheet is provided (Catalog # ARY022B).
2.15. In Vitro Chemotaxis of CPCs
CPCs-PL migration was investigated by Boyden chamber assay using serum-free medium as negative control and ACs-CM pre-treated for 24 h with IL-1β (PeproTech, Rocky Hill, NJ, USA), 5% PL or both stimuli as chemoattractants (see Section 2.13
for CM preparation). Cells were plated at a density of 12 × 104
/chamber on the top of the filter inserts and incubated for 4 h at 37 °C with 5% CO2
. Cells migrated to the lower surface of the filters were fixed in ethanol, stained with toluidine blue and quantified by a bright field microscope (Leica DMi1; Leica Microsystems, Wetzlar, Germany). Data were reported as fold-increase migration, which represented migration extent of CPCs in each condition referred to the control (migration under the influence of conditioned media from non-treated ACs) set to 1 value. Each experiment was performed in triplicate and repeated at least three times (N
2.16. In Vitro Scratch Assay
ACs at passage 1 were plated in 6-well plates, cultured until confluence and treated with either 10% FBS or 5% PL for 24 h. In parallel, CPCs-PL were cultured until confluence. Cell monolayers were washed extensively with PBS to remove residual of factors, scratched using 100 µL pipette tips and covered with serum-free DMEM HG culture medium. Scratch closure was monitored from t0
= 0 h to t1
= 24 h and t2
= 48 h with inverted microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany). Analysis of acquired images was performed with TScratch software (https://github.com/cselab/TScratch
) as reported by Romaldini et al. [15
]. Experiments were performed in triplicate on three different primary cultures (N
2.17. Statistical Analysis
All data are presented as means and standard deviation (SD) or standard error of the mean (SEM) when based on the mean of duplicates or triplicates. Normal distribution of values was assessed by the Shapiro–Wilk normality test. Unpaired Student’s t-test was used to determine statistical significance for normally distributed data and Mann–Whitney test in the absence of a normal distribution to compare ACs-PL versus ACs-FBS or ACs-PL versus CPCs-PL. Level of significance was set at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Kruskal-Wallis with Dunn’s multiple comparison test was used to analyse in vitro scratch assay and one-way ANOVA for in vitro chemotaxis assay followed by post-hoc Sidak’s multiple comparisons test for group differences. Data were analyzed with GraphPad Prism® 8.0 software (GraphPad Software, Inc., San Diego, CA, USA).
Given the therapeutic use of platelet derivatives in orthopaedics, the aim of our study was to explore their stimulatory activity on in vitro cartilage cell homeostasis, focusing on chondro-progenitor cells (CPCs). Hence, in vivo setting was mimed by performing cartilage explant cultures derived from human biopsies in PL- or FBS-supplemented medium. In particular, this study sustains that articular cartilage from joints of patients with ongoing OA contains chondro-progenitor cell population that can be mobilized and subsequently ex vivo recruited from intact tissue by treatment with PL. Conversely, no cell population was recruited when cartilage chips were cultured in medium with FBS. This is the reason why a direct comparison between CPCs-FBS and CPCs-PL was missed in order to investigate the biology of this cell type.
Several studies have already confirmed the presence of stem/progenitor cells in articular cartilage and revealed that their outgrowth needs to be induced as occurs in pathologic condition or by application of external stimuli and trauma [56
]. PL showed to be a powerful stimulus in this context since it is a cocktail of platelet released-growth factors, cytokines and chemokines—i.e., PDGF, IGF or SDF-1α (CXCL12)—widely studied for their multiple effects in the field of regenerative medicine, including chemoattraction of human progenitor cells [59
In order to identify distinctive traits of CPCs among mature cartilage cell population, we compared cells isolated from cartilage chips cultured in presence of PL (CPCs-PL) with ACs enzymatically released from the surrounding tissue and expanded in PL. In parallel, we considered also the comparison between the latter and the AC counterparts maintained in culture with FBS.
Due to the high content in mitogenic growth factors, platelet products exert a strong proliferative stimulus on several in vitro cell cultures, as previously reported [14
]. In this study, the proliferative trend demonstrated by doubling number and cyclin D1 protein levels were markedly higher in ACs-PL compared to ACs-FBS and similar between ACs-PL and CPCs-PL. These results are in accordance with previous works, where PL treatment on partially growth-arrested chondrocytes is reported as responsible for their cell cycle re-entry and strong mitogenic response [14
]. Moreover, we further showed that the increase in proliferation is concomitant with a senescence attenuation in ACs-PL, at a similar level to that of CPCs-PL. In general, a subset of senescent cells is characterized by cell cycle irreversible arrest; meanwhile, quiescent and slowly proliferating cells can restart or increase proliferation after mitogenic stimulation [62
]. Thus, our results reconfirm that PL is able to induce the re-entry of quiescent ACs into the cell cycle progression with consequent decrease of the senescent cell fraction no longer able to replicate. However, a recent hypothesis suggested senescence as a not-terminal cell event [64
], thus platelet derivatives could even induce reversion from a senescent state. Indeed, it was shown that PRP is responsible for senescence recovery as well as induction of stem cell re-proliferation and differentiation in aged mice [65
]. In general, our findings sustain that PL is suitable for expansion of chondrocytes intended for therapeutic applications, especially since senescent cartilage cells are involved in the development of OA in joints and their clearance may attenuate its progression promoting a pro-regenerative milieu [66
Presence of MSC-related surface markers in CPCs supports their progenitor nature as described in previous reports [36
]; meanwhile, their expression in ACs was due to phenotypic change known as dedifferentiation during monolayer culture [41
]. CPCs-PL exhibited lower expression of CD106 than ACs-PL, which is in line with the results reported in a recent study [68
] showing negativity for this chondrocyte marker in MSCs-like progenitors isolated from OA cartilage. Interestingly, CPCs-PL were consistently positive for CD166, whose co-expression with CD105 was reported in both bone-marrow-derived and cartilage mesenchymal progenitor cells [10
]. Since the percentage of CD166+
cells increased in ACs-PL compared to ACs-FBS and results were similar to those found in CPCs, it suggests that PL is able to bring mature ACs to an earlier differentiation stage.
Aspects mostly highlighting the progenitor nature of CPCs-PL in this work were both their clonogenic potential and nestin expression. Interestingly, enzymatically digested ACs, unable to form colonies, acquired this ability when they were switched from FBS- to PL-supplemented medium. Articular cartilage contains a subpopulation of self-renewing cells, mainly concentrated in the superficial layer [9
], but it was also reported that cells with stem cell phenotype can emerge even from fully differentiated chondrocytes [69
]. Despite ACs used in this study being quiescent and mature, PL stimulation seemed to bring out certain features of stemness.
Nestin is a cytoskeletal protein of type VI intermediate filaments known to be expressed in proliferating and migrating stem/progenitor cell subsets in several human tissues [43
], both during embryonic development [70
] and after injuries in adulthood [26
]. Our results are in line with the study of Fellows et al. [71
], which detected nestin-labelled cells in human articular cartilage. An upregulation of this marker was detected in CPCs-PL compared to the weak expression observed in ACs-PL. However, self-renewing cells in colony units of ACs-PL were positive to nestin as well as in CPCs-PL. Nestin-expressing cells compose a quiescent reserve in adults that, if properly reactivated, is able to proliferate, differentiate and migrate. These events are triggered after injury, implying the involvement of these cells in processes of tissue regeneration [26
], although the precise mechanisms have not been understood yet. Therefore, given the involvement of PL in such events, including the transient activation of the inflammatory cascade during tissue regeneration, some of its described effects could be due to activation and amplification of a subset of nestin-positive cells within the cartilage. Since CPCs-PL and ACs-PL were very similar in terms of proliferation, senescence levels and potential to form colonies, our findings suggest that PL retrieved a mixed cell population from cartilage tissue enriched in chondro-progenitors among committed cells.
Considering the features of CPCs-PL, which showed high proliferation capacity and stem/progenitor behaviour, we analysed their chondrogenic potential in view of a future therapeutic application. Generally, adult chondrocytes are characterized by a finite capability to form stable cartilage in vivo, that is gradually lost during in vitro monolayer culture [72
]. ACs-PL displayed very low expression of type II collagen and a similar level of type I collagen in comparison to ACs-FBS, as previously reported [14
]. CPCs-PL showed instead a slight but statistically significant increase in type II collagen expression compared to ACs-PL and a similar level of type I collagen. Such a trend in collagen distribution among cells considered chondro-progenitors is also confirmed by previous works [56
]. Together with this change in collagen levels, a statistically significant difference was also found in the expression of the master regulator of chondrogenesis SOX9
] among the three experimental groups, with a decrease in ACs-PL compared to ACs-FBS and a maintenance in CPCs-PL compared to ACs-PL. Indeed, it has been previously reported that chondro-progenitors showed decreased SOX9
expression compared to fully differentiated chondrocytes, but it is maintained over extended monolayer culture passages together with chondrogenic potential [74
]. Finally, Pereira et al. [14
] have already demonstrated that PL, contrary to animal supplement, supports chondrocyte expansion preserving their chondrogenic phenotype both in vitro and in vivo.
In agreement with previous reports [36
], CPCs-PL, when properly induced, underwent chondrogenic, osteogenic and adipogenic differentiation, capabilities not usual in articular cartilage. Such a trilineage differentiation potency is a typical property of adult MSCs, and thus it could be considered a distinctive trait of CPCs compared to normal chondrocytes, although adipogenesis is controversial and sometimes reported as limited or not inducible in these cells [57
]. However, ACs-FBS and ACs-PL, derived from enzymatically digested tissue in toto, showed trilineage differentiation potency in our hands, probably because these two populations already contain the subset of progenitor/stem cells, as also reported by Alsalameh et al. [12
In orthopaedics, several trials have been conducted to test the potential application of platelet derivatives, although their contributions still need to be clarified. Proteomic studies have shown a plethora of protein-based bioactive factors contained in platelet products [76
]. Among them, released growth factors, such as TGF-β, PDGF, IGF, and bFGF (basic fibroblast growth factor), from platelets were shown to promote cartilage matrix synthesis and chondrogenic potential [78
The strategies for progenitor cell recruitment/enrichment could be exploited for in situ reparative therapies based on platelet products, as previously demonstrated by Siclari et al. [20
], or in a conventional tissue engineering approach since CPCs have shown multiple advantages compared to ACs and even to BM-MSCs for scaffold-assisted cartilage regeneration, as recently reported by [79
]. In this perspective, we reported ectopic cartilage formation after implantation of expanded PL-recruited progenitors (CPCs-PL) in nude mice. Neo-formed tissue showed a typical metachromatic staining, and it was type II collagen positive and type X collagen negative and thus ascribable to a hyaline-like cartilage without hypertrophy signs. Therefore, migrating cells from articular cartilage under PL treatment (CPCs-PL) can be further considered a population consisting of chondro-progenitors since they were shown to be preferentially committed towards chondrogenic rather than osteogenic lineage in vivo.
Characterization of the conditioned media from ACs-FBS, ACs-PL and CPCs-PL could help in understanding the effects of PL on cartilage-derived cells and its involvement in tissue dynamics. Indeed, human chondrocytes, both normal and osteoarthritic, produce chemokines and express a variety of chemokine receptors, suggesting that their autocrine/paracrine pathway within cartilage may be involved in its homeostasis and matrix remodelling [55
]. Secretome derived from digested ACs contains a plethora of molecules, including not only chondrocyte factors but also angiogenic proteins, pro-inflammatory cytokines, hypertrophic differentiation markers and chemokines with chemoattractant activity on immune system cells. Since cells used in the present study derived from articular cartilage biopsies with ongoing inflammatory processes, the described secretory profile could reflect the long-lasting exposure to a pathological joint environment.
PL treatment induced increased release or de novo secretion of some pro-inflammatory cytokines and chemokines by ACs. Indeed, it is well known from previous works that PL exerts an initial strong pro-inflammatory activity resulting in NF-κB activation and secretion of pro-inflammatory cytokines, events that are transient in order to prime certain defensive and reparative tissue mechanisms, then it inhibits and promotes resolution of the inflammatory phase [14
Interestingly, the secretory profile of CPCs-PL differed from that of ACs-PL for soluble components involved in bone and cartilage biology or correlated with hypertrophic phenotype. Among them, the Wnt pathway antagonist Dkk-1 was highly upregulated in CPCs-PL. Dkk-1 is highly expressed in cartilage, and it is required for chondrogenic differentiation of MSCs, redifferentiation and prevention of hypertrophy of chondrocytes [80
]. Furthermore, a low amount of ST2 was also detected in CPCs-PL compared to ACs-PL. ST2 has recently been described as a chondrocyte differentiation regulator that promotes expression of hypertrophic markers after RUNX2 induction [50
]. Finally, osteopontin (OPN), a protein that contributes to hypertrophic phenotype in chondrocytes [81
], was lower in the CM of CPCs-PL compared to the CM of ACs-PL. Another advantage of CPCs-PL over ACs-PL is that the former cell type secreted higher amounts of CST3, an inhibitor of cathepsins. In OA cartilage with severe lesions, a reduced inhibitory activity of CST3 has been reported, suggesting that downregulation of CST3 contributes to articular cartilage damage [82
]. DPPIV release was also significantly higher in CPCs-PL than in ACs-PL. In a murine model of collagen-induced arthritis (CIA), it has been demonstrated that DPPIV injection decreases the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue [83
Moreover, PL markedly increased the release of CCL5 (RANTES) by both ACs-PL and CPCs-PL, suggesting that PL exposure may promote the migration of joint resident cells towards damaged areas where they could engraft and take part in regenerative processes, as previously observed in degenerated intervertebral disk, where annulus fibrosus cells were mobilized in response to this secreted chemokine [84
]. CCL5 has been reported to be involved also in MSCs recruitment [54
], and thus it is considered attractive for regenerative therapies. As already reported [85
], high levels of CXCL12/SDF-1α in chondro-progenitor secretome may indicate their ability to mobilize other endogenous progenitor cells within injured articular joints and recruit immune cells in order to help in mediating tissue repair.
A recent study has confirmed that chondro-progenitors are more suitable candidates for therapeutic treatment of meniscus injury than BM-MSCs since they resisted to hypertrophic differentiation during tissue repair [86
]. Similarly, and according with our findings, we can suggest that PL-induced activation of such CPC population able to counteract cell hypertrophy within articular cartilage could be considered beneficial in therapeutic treatments for OA. However, further and more focused studies will be needed to investigate our hypothesis.
Although their role in cartilage disorder (OA) progression has not yet been clarified, chondro-progenitor cells has shown to be responsive to injury. In this study, we demonstrated that they are able not only to release into the surrounding environment chemoattractant factors but also to actively migrate in response to signals coming from inflamed chondrocytes. This is in accordance with the demonstrated capability of CPCs to migrate across the articular surface to sites that have suffered trauma and therefore display an ongoing inflammation [57
]. These findings suggest that these mobilized cells may organize a kind of regenerative response in an attempt to restore the perturbed cartilage homeostasis in a damaged joint. Finally, PL exerted a general strong chemotactic action on the entire cartilage cell population, including mature chondrocytes (ACs) that usually are considered not provided with a mobile phenotype. Conversely to culture with FBS supplemented medium, ACs exposed to PL, probably as a result of changes in cell morphology and cytoskeletal rearrangements, acquired enhanced motility and were able to close a wound scratch in vitro, suggesting the possibility of using PL to promote their involvement in the overall tissue reparative process.