Periosteum-Derived Mesenchymal Stem Cells Secretome - Cell-Free Strategy for Endogenous Bone Regeneration: Proteomic Analysis in Vitro

Objectives: Millions of people worldwide are affected by diseases or injuries which lead to bone/tooth loss and defects. While such clinical situations are daily practice in most of the hospitals, the widely used treatment methods still have disadvantages. Therefore, this field of medicine is actively searching new tissue regeneration techniques, one of which could be stem cell secretome. Thus, the purpose of this research study was to perform the detail proteomic analysis of periosteum-derived mesenchymal stem cells secretome in order to evaluate if it is capable to induce osteo-regenerative process.

Material and Methods: Periosteum-derived mesenchymal stem cells (PMSCs) were extracted from adult male New Zealand White rabbits. Cells were characterised by evaluating their differentiation potential. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed.

Results: PMSCs were extracted from adult male New Zealand White rabbits. In order to characterise the extracted PMSCs, they were differentiated in the directions which mainly describes MSC multipotency - osteogenic, myogenic and adipogenic. A total of 146 proteins were detected. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed. The resulting protein composition indicates the ability to promote bone regeneration to fully mature bone.

Conclusions: Bioactive molecules detected in periosteum-derived mesenchymal stem cells secretome initiates the processes required for the formation of a fully functional bone.

J Oral Maxillofac Res 2021;12(2):e2

doi: 10.5037/jomr.2021.12202

Accepted for publication: 22 June 2021

Keywords: bone regeneration; mesenchymal stem cells; periosteum.

INTRODUCTION

Periodontal diseases and face injuries have led to bone and tooth loss and defects, which have become a global concern, often affecting the health and quality of life of the entire population and placing a heavy financial encumbrance on community [1,2]. Such bone defects regeneration can be defined as a complex mechanism based on the interaction between osteogenic, angiogenic, chondrogenic, etc processes able to drive bone growth and tissue restoration [3-5]. During bone defect regeneration process, different cell lineages interact with each other in order to promote tissue healing. In novel bone development, osteogenic, angiogenic, and neurogenic processes are closely connected [6,7]. The blood vessels of bone tissue can transport minerals and growth factors and, at the same time, represent the physical structures around which bone deposition start [8,9].

Over the past decade, there has been a growing interest in the therapeutic application of autologous products/stimulants such as platelet rich fibrin (PRF), platelet rich plasma (PRP), plasma rich in growth factors (PRGF), mesenchymal stem cells (MSCs) etc for the regeneration/treatment of bone defects [10-12]. For multipotency and ability to differentiate into osteogenic cell lineage, stem cell-based therapy is assessed as one of the most perspective technique in bone regenerative medicine [13]. Even though cell-based therapies including injection or transplantation of MSCs are promising strategies, some concerns remain, such as technical limitations and low survival rates of transplanted cells [14]. Furthermore, some studies report increase in apoptosis after transplantation often triggers an immune response, resulting in worsening of the diseased condition or rejection of the transplanted cells [15,16]. Recent studies revealed that bone marrow, stromal, hematopoietic MSCs can contribute to tissue regeneration not only through their multipotency but also by stimulating the recipient cells via paracrine mechanisms [17,18]. The paracrine effects are mediated by secretomes including cytokines and chemokines. As the secretomes from MSCs contain various factors exerting several biological effects, they are also expected to be applied clinically and provide novel strategies for regenerative medicine [19]. However, according to the studies different types of MSCs produces and secrets different bioactive molecules. For example, secretomes obtained of human brown and white adipogenic MSCs has different protein composition and according to that greater abundance of immunoreactive proteins were detected at in the secretome from brown adipogenic MSCs secretome compared to white adipogenic MSCs secretome [20]. Therefore, it was demonstrated that the origin of MSCs determined the proteomic profile of MSCs secretomes and predetermined it’s biological functions [21].

The origin of the MSCs not only depends the specific molecules which secret the cell, but also determines their differentiation potential [22,23]. For example, MSCs isolated from adipose tissue will more easily differentiate in the adipose direction compared to bone marrow derived MSCs and vice versa [24]. Meanwhile, osteogenesis would be more easily induced in MSCs derived from bone-adjacent tissues, such as periosteum. For this reason and secretome produced by periosteal-derived MSCs (PMSCs) may be well suited for bone regeneration. However, protein composition and biological functions of PMSCs secretome has not been fully investigated yet.

Thus, in this research study, we aimed to characterize periosteum-derived mesenchymal stem cells secretome, in terms of their proteomic composition.

MATERIAL AND METHODS

Cell source

PMSCs were extracted from three randomly chosen adult male New Zealand White rabbits, weighing approximately 3 kg. Rabbits were used in this study with the approval of the State Food and Veterinary Service (identification code: G2-55). The study was conducted from February 1, 2017 to December 31, 2019. The rabbits were housed in a temperature-controlled room (21 to 23˚C) and accommodated under a 12 h light-dark cycle. An individual cage was intended, and animal was fed by standard dried diet and water ad libitum. The premedication was induced by injection of acepromazine (0.5 mg/kg) (Temprace, Oudewater, Netherlands) in thigh muscles and a subcutaneous injection of buprenorphine (0.03 mg/kg) (INDIVIOR INC, North Chesterfield, USA). General anaesthesia was achieved by injection of ketamine hydrochloride (35 mg/kg) (Salfarm Danmark A/S, Kolding, Denmark) and xylazine hydrochloride (5 mg/kg) (Xylomed Pharmaceuticals Limited, Gloucestershire, UK) in thigh muscles. The Carbomer Eye Gel (Oftagel^® 2.5 mg/g - SANTEN OY; Tampere, Finland) was used to keep the eyes wet. Surgical procedure was performed using a special warming surgical table and special cover to keep the animals warm and achieve better sterility. After shaven of the calvaria area, it was disinfected with alcohol and local anaesthetic with articainum/epinephrinum performed (Ubistesin™ forte [40 mg + 5 mcg/ml] - 3M Deutschland GmbH; Neuss, Germany). After preparation for operation, the surgical area was expanded using a sagittal incision through skin and periosteum around the entire thickness. A 5 x 5 mm of periosteum was cut out.

Isolation and cultivation of PMSCs

PMSCs were isolated from periosteum obtained from the calvaria site, as previously described [25]. Briefly, tissue was further processed under sterile laminar flow conditions. Using sterile scizors tissue was minced into smaller pieces, approximately 1 mm³ in size. Periosteum tissue pieces were transferred to the sterile 15 mL vial with 4 ml of 1 mg/ml collagenase A solution prepared in DMEM for 18 hours at 37 °C while gently stirring. Then, tissue pieces were separated from the cells by filtrating through a sterile 70 µm nylon mesh sieve. The cell suspension was centrifuged at 400 g for 10 min at 4 °C, the supernatant was then discarded, and cells were suspended in growth media (GM) composed of Dulbeccos Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and antibiotics: penicillin 100 U/mL, streptomycin 100 mg/mL. PMSCs were sowed at 75 cm² Falcon flasks, at the density of 40,000 cells/cm². Later it was grown in GM. All growth supplements were purchased from Cambrex Bio Science Walkersville, Inc. (Walkersville, Maryland, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Subconfluent rPMSCs were trypsinized and used in subsequent experiments.

Evaluation of PMSC differentiation potential

In order to evaluate PMSCs differentiation potential, cells were grown in adipogenic, myogenic and osteogenic differentiation inducing media (Figure 1). For adipogenic differentiation induction, cells were grown in DMEM supplemented with 10% FBS, 1% penicillin streptomycin solution (Sigma-Aldrich Co.; Darmstadt, Germany), 1% glutamine (Sigma-Aldrich Co.; Darmstadt, Germany), 1 µM dexamethasone (Sigma-Aldrich Co.), 1 µM indomethacin (Sigma-Aldrich Co.; Darmstadt, Germany), 500 µM 3-isobutyl-1-methylxanthine and 10 µg/ml human recombinant insulin. Cells were grown in this media for 4 days. Then, cells were fixed with 4% formaldehyde for 10 min at room temperature. Latter fixed cells were washed with distilled water followed by 5 min washing with 60% isopropanol at room temperature. Lipid reserves stored in the cells were stained with Oil Red O staining solution (0.5% solution in isopropanol - Sigma-Aldrich Co, Darmstadt, Germany) for 10 to 15 min. After removing the dye, cells were washed with deionized water until the water becomes clear. Red stained droplets were visualized and captured with a charge-coupled device (CCD) camera (EXi Blue™ - QImaging; Surrey, British Columbia, Canada) attached to inverted phase contrast microscope (Olympus IX51 - Olympus Co.; Tokyo, Japan). For myogenic differentiation induction, cells were grown in DMEM supplemented with 2% horse serum and 1% penicillin streptomycin solution, for one week. Cell differentiation was evaluated by visualizing multinuclear cells with Crystal violet staining. Briefly, myogenic differentiation media was removed, cells 3 times washed with PBS, and stained with 0.1% Crystal violet solution for 30 min (prepared in 20% ethanol; Sigma-Aldrich Co.). Then cells were washed with distilled water and multinuclear cells were visualized and captured with a CCD camera (EXi Blue™ - QImaging) attached to a microscope (Olympus IX51 - Olympus Co.). For osteogenic differentiation cells were grown in DMEM supplemented with 10% FBS, 1% penicillin streptomycin solution, 10 mM β-glycerophosphate, 25 µg/ml ascorbic acid and 50 nM dexamethasone. Differentiation was carried out for 21 days, half of the media was replaced every two/three days. Osteogenic differentiation was confirmed by staining formed calcified extracellular matrix (ECM) with alizarin red S (ARS) dye. Osteogenic differentiation media was removed and cells were fixed with 4% formaldehyde for 10 min at room temperature. Then cells were washed 3 times with PBS and stained with 2% ARS (pH 4.1 - 4.2). Cells formed calcified ECM (stained in red) were visualized and captured with a CCD camera (EXi Blue™ - QImaging) attached to a microscope (Olympus IX51 - Olympus Co.).

Secretome preparation

PMSCs were seeded at the density of 40 000 cells/cm² in the 75 cm² flasks (Thermo Fisher Scientific, Inc., Waltham, USA). The next day, growth media was removed, cells were 3 times washed with PBS (Gibco™ - Thermo Fisher Scientific, Inc.) and serum-free DMEM was added. Cells were grown for 3 days in 37 °C with 5% CO₂ atmosphere and 95% humidity. After predetermined time the secretomes was collected to 50 ml tubes (Thermo Fisher Scientific, Inc.) and centrifuged for 15 min at 6000 RCF. Supernatant was filtered through 0.22 µm syringe driven PVDF filter (Thermo Fisher Scientific, Inc.) to new 50 ml tubes, and stored at 4 °C. All secretomes were used up in 30 days after collection.

Proteomic analysis

Sample preparation

Filter aided sample preparation (FASP) [26] method was used for protein digestion prior to mass spectrometry analyses. Protein lysates were processed by the FASP using Microcon^® 30k centrifugal ultrafiltration units (Merck Millipore; Darmstadt, Germany) operated at 10,000 g. Briefly, the sample was diluted with 200 μL of 8 M urea (pH 8.5), placed in a filter unit, centrifuged and washed two times with 100 μL of 8 M urea. Then, 100 μL of 55 mM iodoacetamide was added to the filters, and samples were incubated for 20 min. Filters were washed twice with 100 μL of 8 M urea followed by two washes with 100 μL of 50 mM NH₄HCO₃ pH 8.0. Protein digestion was then performed by adding trypsin in 50 μL of 50 mM NH₄HCO₃ at an enzyme to protein ratio of 1:100 and incubating overnight at 37 °C. Peptides were collected from the concentrators by centrifugation at 10000 g for 10 min and additionally eluted using 20% CH₃CN. The eluates were combined, acidified with 10% CF₃COOH and peptides were dried in a speed vacuum for 2 hours at 45 °C. The lyophilized peptides were redissolved in 0.1% formic acid.

LC-MS based protein identification

Liquid chromatographic (LC) analysis was performed in a Waters^® Acquity^® Ultra Performance LC system (Waters Co., Wilmslow, Manchester, UK). Peptide separation was performed on an Acquity^® UPLC HSS T3 250 mm analytical column (Waters Co.). Data were acquired using Synapt G2 mass spectrometer (MS) and Masslynx^® version 4.1 software (Waters Co.) in positive ion mode using data-independent acquisition (UDMSE). The capillary voltage was set at 2.8 kV, and the source temperature was set at 80 °C. Scan time was set at 0.75 s. Raw data were lock mass-corrected using the doubly charged ion of (Glu1)-fibrinopeptide B (m/z 785.8426; [M+2H]2+). Raw data files were processed and searched using ProteinLynx Global SERVER™ (PLGS) version 3.0.1 (Waters Co.). Data was analysed using trypsin as the cleavage protease, one missed cleavage was allowed, and fixed modification was set to carbamidomethylation of cysteines, variable modification was set to oxidation of methionine. Minimum identification criteria included 1 fragment ions per peptide, 3 fragment ions and one peptide per protein. The following parameters were used to generate peak lists:

• Low energy threshold was set to 150 counts;

• Elevated energy threshold was set to 50 counts;

• Intensity threshold was set to 750 counts.

UniprotKB/SwissProt database (www.uniprot.org/) were used for protein identification. The PANTHER classification system (www.pantherdb.org/) was used for GO mapping and functional annotation of proteins. Proteins were sorted by inducible biological function and plotted in pie charts and tables describing specific protein functions.

Statistical analysis

Results were processed by using Microsoft Office Excel 2021 software (Microsoft Corporation, Redmond, Washington, USA). All proteomic results are presented as pie charts from three independent experiments (N ≥ 3 samples per group).

RESULTS

PMSCs characterisation

In order to characterise the extracted PMSCs, they were differentiated in the directions which mainly describes MSC multipotency - osteogenic, myogenic and adipogenic. It was observed that osteogenic differentiation induction induces PMSC accumulate calcium phosphate deposits in their ECM (Figure 1B). Myogenic differentiation analysis revealed that PMSCs are capable to form multinucleated cells as well (Figure 1D). Finally, adipogenic differentiation results showed lipid droplets on the cell monolayer, which is the characteristic of adipocytes (Figure 1F).

Proteomic analysis of PMSCs secretome

Further, the detail proteomic analysis of PMSCs secretomes was performed. A total of 146 proteins were detected (detail protein list is depicted in Appendix 1). Among them 55 (38%) were determined as ECM proteins and 91 (62%) were assigned as proteins belonging to other cellular regions (cellular proteins) (Figure 2A).

All determined proteins were further analysed in PHANTER classification system to determine their GO biological function. Results showed that proteins in PMSCs secretome contributes to biological processes associated with osteogenesis, cell-ECM interaction, chondrogenesis, cytoskeleton, differentiation, ECM formation, immune response, metabolism, migration, neurogenesis, signalling, transport, wound healing, and angiogenesis (Figure 2B).

To understand better how PMSCs secretome can stimulate osteo-regeneration process, the detail analysis of proteins, which were linked to immune response, osteo, angio, and neurogenesis biological functions, was performed. The resulting protein composition indicates the ability to promote bone regeneration to fully mature bone. The proteins found induce osteogenesis, angiogenesis and immune response. Many of them also have a synergistic effect that promotes the activities of other processes mentioned above. Specific proteins and their function in theses biological processes are listed in Tables 1 - 4.

DISCUSSION

Bones fractures especially in maxillofacial area or face asymmetries caused by traumas, violence, cancer excisions or other surgical treatment are exceptional because, unlike disorders in other areas, this condition is accompanied by psychosocial changes in a person’s quality of life [27]. Due to the non-physiological asymmetries and face proportions, sudden changes in the appearance of the face, a person will experience stress, because of this it is necessary to reconstruct facial tissues and return patients’ fulfilling life and self-confidence. Depending on the size of the population in the countries, facial fractures occur from a few hundred to several thousand cases per year [28]. Thus, skin, bone, cartilage reconstruction procedures are a daily clinical practice in most of the hospitals. However, widely used treatment methods still have shortcomings and this field of medicine is in the ongoing search stage for new and modern tissue regeneration techniques. Many ways have been tried to promote or accelerate better tissues healing but still there is no gold standard or reliable method in clinical practice. Stem cells have been thought to be the future of regenerative medicine, but due to poor and unstable clinical results and the existing regulation of bioethical organizations, this field of regenerative medicine is becoming less relevant [29]. Other methodologies such as blood concentrates or growth factors, have been used in clinical practice, however, it did not find a wide audience due to bioethical deficiency or small and doubtful clinical effect [10]. In recent years, cell free therapy may seem appropriate for more accurate and faster regeneration of many tissues but still needs to be better investigated in various aspects [17-19,30,31].

Secretomes may be perfect for cell-free therapy in the treatment of gastrointestinal diseases, cancers, skin diseases, heart ischemic diseases, autoimmune disorders, can help for Covid-19 infected patients, and etc. [32-34]. However, there are very few clinical trials in this area and they have been conducted recently. Kshitiz et al. [35] used secretome of bone marrow-derived stromal cells to revealed a cardioprotective biochemical cocktail, Jarmalavičiute et al. [36] reported that secretome obtained from human dental pulp MSCs were able to reduce apoptosis of neurons. There are many successful experiments which leaded to further trials. Moreover, it is also known that the composition of bioactive secretome molecules depends on the type and origin of the cells [22,23,37]. In the natural environment (i.e., natural tissue) cells use these molecules to communicate with each other. By sensing each other’s expressed molecules cells know how to behave [38]. For example, in the case of bone fracture, osteoblast secreted signalling molecules composition alters. This change is further recognized by MSCs reside in surrounding tissues. These stem cells start to migrate to the damage site, were they start to differentiate into the osteoblasts. In parallel, these MSCs continue to synthesize and express their signalling molecules which is further recognized by other cells - this ensures a successful osteo-regenerative process [7,39]. Thus, as cells use the secreted molecules to communicate with each other, it is likely that cytokines, chemokines and various growth factors produced by MSCs derived from tissue close to bone should best stimulate bone regeneration. The periosteum is a tissue which is constantly connected to the bone. It performs transformation of bone tissue. As a consequence, it contains many stem cells that migrate to the site of injury in the event of bone damage and count its regeneration [40,41]. Moreover, studies have shown that PMSCs are not only multipotent (as was also confirmed by our results), but also tend to spontaneously differentiate in the osteogenic direction [17,42]. Therefore, the secretome of PMSCs should be suitable as a cell-free strategy for bone regeneration. However, the detailed protein composition and biological functions of PMSCs secretome has not been fully investigated yet. Thus, in the current study, we aimed to characterize it.

After detail proteomic analysis it was determined that PMSCs secretome is rich in proteins which is known to stimulate osteogenesis. Among the detected proteins which were related with osteogenesis, most of them were associated with bone-specific ECM formation (fibrillin-1, fibrillin-2 etc.). It is well known, that the successful MSC differentiation to osteoblasts can only occur then bone-specific ECM is forming [43]. Therefore, the initiation of such a process is important to ensure damaged bone regeneration.

During new bone formation not only osteogenesis but also angiogenesis, neurogenesis and even immune response are important [3,44]. In the beginning of bone fracture healing process, to the damage tissue site various immune system molecules (neutrophils, monocytes, macrophages) are attracted. They not only remove necrotic cells and damage bone fragments, but also begin to secrete various inflammatory, chemotaxic and progenitor mediators (e.g. stromal derived factor-1α, tumour necrosis factor α, interleukin-1β, interleukin-6, chemokine ligand 2, bone morphogenetic protein, fibroblast growth factor, WNT family proteins) in order to attract MSCs from bone marrow, periosteum or cortical bone to site of the lesion [45]. Thus, immune response stimulation is essential for the beginning of bone regeneration process. Our results showed that PMSCs secretome is also rich in various proteins which can stimulate these immune system molecules. For example, prosaposin, annexin A1, alpha-2-macroglobulin, plasminogen activator inhibitor 2, etc.

Neurogensis is also particularly important for fully functional new bone formation. Only with the formation of a complete neural network in the bone will complete homeostasis of this tissue be ensured [46]. Proteomic analysis revealed neurogenesis-promoting proteins as follows: collagen alpha-1(IV) chain, calreticulin, etc.

Angiogenesis can be called one of the most important factors in the process of bone regeneration. During this process, different cell lines interact with each other, thus promoting tissue healing. In the formation of new bone tissue, osteogenic and angiogenic processes are closely related. The blood vessels in the bone tissue can carry minerals, growth factors, and also play a role in the physical structures around which bone deposition begins [3,4,14]. Proteomic analysis of proteins that promote angiogenesis, wound healing, and osteogenesis include thrombospondin-1, collagen alpha-1(III) chain, keratin, type II cytoskeletal 1, etc.

Finally, it is important to mention, that we have found that many proteins which were detected in PMSCs secretomes had inherent and overlapping functions. E.g.,annexin A2 is involved in immune response and osteogenesis or annexin A1 is involved in angiogenesis and immune response. However, it is known from the other studies that the role of a protein may depend on the cell that synthesizes it. And in our case, when annexins A1 and A2 are synthesized by PMSCs then their functions are to induce angiogenesis or form a new bone, but not to stimulate an immune response [20]. Furthermore, the detected proteins biological functions overlapping could also appeared because most of these proteins are not the main initiators of all those established processes [47,48]. For example, galectin-3. This protein belongs to lectin family and as known from the literature it can be involved in many different signaling pathways. It demonstrates pro-inflammatory properties by recruiting neutrophils and other immune cells to the infected sites [49]. It interacts with integrin receptors and in this way mediates cell apoptosis. It also can co-operate with various ECM proteins (collagen IV, elastin, vitronectin, etc.) and thus affect cell adhesion process [50].

CONCLUSIONS

In this study for the first time the detailed proteomic analysis of periosteum-derived mesenchymal stem cell secretome was performed. Obtained results show that cytokines, chemokines, and growth factors detected in periosteum-derived mesenchymal stem cells secretome initiates the processes required for the formation of a fully functional bone. Therefore, periosteum-derived mesenchymal stem cells secretome can be used as potential, new and innovative cell-free bone regeneration technique.

APPENDIX 1

ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

The authors declare no conflict of interest related to this study.

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