Recellularization of Canine Placental Extracellular Matrix: Mesenchymal Stem Cells Applied to Tissue Bioengineering

ECM: Extracellular Matrix

VEGF: Vascular Endothelial Growth Factor

MSCs: Mesenchymal Stem Cells

CBMC: Canine Bone Marrow

CDPC: Canine Dental Pulp

SDS: Sodium Dodecyl Sulfate

PBS: Phosphate Vuffered Saline

UV: Ultraviolet Light

α-MEM: Minimum Essential Medium Eagle-Alpha Modification

FBS: Fetal Bovine Serum

SEM: Scanning Electron Microscope

Biological biomaterials obtained from organs and tissues decellularization techniques have received expansive emphasis on tissue bioengineering [1-5]. The decellularization aims remove the cells from the tissue and maintain the three-dimensional structure of your extracellular matrix and their bioactivity, resulting in a acellular biological scaffolds [2,5-8]. 

Acellular biological scaffolds, also known as extracellular matrix (ECM), may be derived from animal species (xenologous origin) and have been widely studied in regenerative medicine [9-14]. 

Usually the use of decellularized biological scaffolds appears as a promising tool in order to eliminate the scarce supply of tissues and organs for transplants [1]. Decellularization of a three dimensional matrix scaffold (i.e., animal placentas) enables the removal of cellular and immunogenic components from an organ/tissue while preserving the ECM with its native micro and macroscopic anatomy [15].

Placentas represents an interesting alternative for obtaining acellular scaffolds to be used in tissue bioengineering [3,10,16]. The characteristics that make the placenta a source of decellularized biomaterial are: high sample supply, rich diversity of extracellular matrix proteins, can be collected without damage to the donor, and a complete defined vascularization [17]. 

Decellularized placentas from different species (humans and animals) including the canine placenta demonstrate be useful as biological scaffolds (tissue or as a hydrogel), corroborating for tissue bioengineering [3,18-23]. Due the ECM properties preserved after removal of cellular content, decellularized placentas support cell migration and proliferation, as well as cell / matrix interaction [3,20,24]. Mouse placental scaffolds cultured with mouse embryonic fibroblasts demonstrated a tri-dimensional repopulation and cell-matrix adhesion [23]. 

Decellularized canine placentas when applied in vitro in interaction with the endothelial progenitor cells derived from the yolk sac transduced with Vascular Endothelial Growth Factor (VEGF), demonstrate interaction with the ECM for recellularization of the tissue [25]. 

In this study, we examined whether the use of decellularized canine placentas to recellularize with Mesenchymal Stem Cells (MSCs) derived from Canine Bone Marrow (CBMC) and Canine Dental Pulp (CDPC) for 7 days in culture. Cell adhesion capacity / matrix interaction was investigated for possible use as a small graft applied to the tissue engineering.

Canine placental decellularization and recellularization 

Placentas of bitches with 35 days of gestation were used. The placentas were Sterile in 0.1% Sodium Dodecyl Sulfate (SDS) for 15 days under stirring in a Sterile environment and washed in Triton X-1001% [20]. The decellularized placentas were passed in a critical point LEICA EM CPD 300, for pre-sterilization of the samples (Figures 1a-1c). They were then washed in 1X Phosphate Buffered Saline (PBS) with 0.5% antibiotic (penicillin-streptomycin) and left in ultraviolet (UV) light for 10 minutes (Figure 1d). The sterility of the decellularized placentas were tested in culture medium with Minimum Essential Medium Eagle with 10% Fetal Bovine Serum (FBS) and in cubated at 37°C with 5% CO2 for 24 hours (Figures 1e and 1f), as standardized by our group [25]. A number of 5 x 104 CBMC and CDPC were plated on non-adherent dishes (Sarstedt) for 7 days in culture (Figure 1g). The cell lines used come from a cell bank standardized by the group [26].

 
Figure 1: Experimental design of canine placenta decellularization and recellularization process.

Immunofluorescence cells and recellularized scaffold 

Non-adherent plates containing samples of placentas recellularized with CBMC / CDPC for 7 days were fixed with 4% paraformaldehyde. Supports were washed with PBS + 0.5% Tween and incubated with the primary antibody fibronectin (Abcamab2413), CD105 (Invitrogen MA5-11854) and CD90 FITC (Abcamab124527) at a dilution of 1: 200, at 37°C for 1 hour. The samples were then washed in PBS + 0.5% Tween and incubated for 1 hour at 4°C with secondary antibody Alexa Fluor 594 (Thermo FisherA30008) and Alexa Fluor 488 (Thermo FisherA-11094). Plates were incubated with DAPI for nuclear staining. They were analyzed in the Confocal Microscope - Olympus Fluo View 1000 (FV1000). 

Scanning electron microscopy 

The decellularized and recellularized placentas were fixed in 4% paraformaldehyde. They were dehydrated in increasing concentrations of alcohol, dried at critical point (LEICA EM CPD 300) and glued with carbon tape in metal (sputter coating) with gold metalizer. Subsequently they were photographed in a Scanning Electron Microscope (SEM) LEO 435VP.

Sterile placental fragments for recellularization assay were performed in 7 days, by suspension in anon-adherent dishes using two types of Mesenchymal Stem Cells (MSC): CBMC and CDPC. The placental scaffolds demonstrated chemotaxis by the cells types, favoring cell/matrix interaction. 

Positive expression for the CD90 marker in the cytoplasm of canine bone marrow mesenchymal stem cells and canine dental pulp cells was demonstrated (Figure 2). 

Figure 2: Immunofluorescence of CBMC and CDPC. A-C: canine bone marrow cells; D-F: canine dental pulp cells (DAPI - cell nuclei; CD90 cytoplasmatic marker). 

By the immunofluorescence assay, it was possible to confirm the adhesion of CBMC and CPDC in the placenta scaffold cultured for 7days (Figure 3). These when in contact with the scaffolds demonstrated high expression of fibronectin (Figures 3c, 3g, 3k and 3o), a structural protein from ECM (preserved in the decellularization process) that helps to the cell/matrix adhesion. In addition, the interaction of the cells with scaffolds demonstrated a positive expression of mesenchymal stem cells markers: CD90+ (Figures 3b, 3d, 3j and 3l) and CD105+ (Figures 3f, 3h, 3n and 3p). 

Figure 3: Immunofluorescence of recellularized canine placental scaffolds with CBMC and CPDC for 7 days in culture. (A, E) and (I, M): DAPI immunostaining (blue) for canine bone marrow cells and canine dental pulp cells, respectively; B and J (CD90) F and N (CD105): Green immunostaining for canine bone marrow cells and canine dental pulp cells, respectively. C, G, K and O: Red immunostaining for fibronectin in the placental extracellular matrix; D, H, L and P: merged. 

The recellularization of canine placenta scaffolds with CBMC and canine bone marrow cells and canine dental pulp cells was also observed by scanning electron microscopy confirming that there was an interaction between the extracellular matrix and cells in culture in vitro for 7 days (Figure 4). Besides MSCs attached to placental ECM scaffolds preserved their collagens structure morphology. 

Figure 4: Scanning Electron Microscopy of scaffolds of recellularized placentas with CMOC and CPDC for 7 days of culture. A and B: canine placenta decellularized fetal and maternal portions, respectively (* observe absence of cell nuclei); C and D: canine placenta recellularized with CBMC; E and F: canine placenta recellularized with CDPC; (→ observe cell / matrix interaction). 

Placentas decellularization techniques of different species have been standardized in recent years. Proteins and the three-dimensional architecture of the extracellular matrix are preserved, applied to recellularization process and used as small fragments tissue or hydrogel for regeneration [17-20,22,23,25,27-29]. 

Previously, the decellularization of canine placentas of 35 days gestation was standardized. In the cellular removal process, we obtained satisfactory results regarding the quantification of the desired gDNA for an acellular biological scaffold. Furthermore, after the decellularization process, canine placentas decellularization demonstrated the preservation of their ECM proteins such as protein structures (collagens type I, III and IV), proteoglycans and adhesion glycoproteins (fibronectin and laminin). Such constitutes ECM molecules directly influence the role of cell adherence to the scaffolds [20]. 

MSCs have been used in regenerative medicine since they are multipotent stem cells with due to their ability to regenerate tissue from mesenchymal origin. Besides, their capability of suppressing immune responses, anti-inflammatory, immunomodulatory and paracrine effects thus reduces transplantation rejection in clinical applications [30]. In addition, Papanikolaou (2019) reviews results in experimental studies using diverse sources of MSC for use in regenerative medicine [31]. 

We cultured bone marrow and dental pulp cells on the 3D decellularized canine placentas scaffolds in non-adherent plaques, according the recellularization process as already observed in the culture with progenitor cells from the canine yolk sac transduced with vascular endothelial growth factor [25], which directly influences the migration / adhesion of cells on the scaffold. 

Mesenchymal stem cells derived from bone marrow has multipotential capacity to differentiate [32]. These, when applied in tissue engineering for regenerative medicine, evidence their role as a pluripotent stem cell because they have the role of bioactive macromolecules [33,34]. 

Against this background, our results showed that the biological scaffold derived from placenta decellularization assay cultured for 7 days with MSCs had positive expression of mesenchymal markers CD90+ and CD105+, demonstrating the cell / matrix interaction in the process of recellularization. These data contributed to the recellularized scaffolds with the MSCs being promising sources for use in regenerative medicine [32,35-38]. 

According to results obtained by human cells derived from bone marrow were efficient in the recellularization process of the small intestine [39], an organ with ECM components similar to canine placenta (collagen, laminin and fibronectin), in addition to the vascularization present in both organs. Cell differentiation in decellularized lungs was observed when cultured for recellularization assay using MSCs derived from mouse bone marrow, suggesting the capacity of these cells to modulate the environments of cells/matrix [40]. 

Moreover, dental pulp stem cells have characteristics similar to MSC, including the auto-renewal capacity and the potential of multiline differentiation [41]. These still have high plasticity, being able to differentiate in neurons, adipocytes and osteoblasts [42] and demonstrate efficacy in therapeutic treatment in dogs with chronic spinal cord injury [26]. DPSC have been widely used as potentially novel therapeutic agents in regenerative medicine due our multipotency and immunosuppressive effects [43]. 

In conclusion we demonstrated that the recellularization of canine placentas with mesenchymal stem cells derived from canine bone marrow and dental pulp were successful. The decellularized placenta samples used in this study for recellularization demonstrated that the native tissue structure preserved ECM components corroborated for cell/matrix interaction. Considering MSCs have been suggested for recellularization in decellularized scaffolds [44], these results provide a basis for the development of new approaches, since mesenchymal stem cells derived from bone marrow and dental pulp may evidence a therapeutic potential for future applications in tissue bioengineering in vivo.

The authors are thankful to Advanced Center of Image Diagnosis (CADI-FMVZ-USP) facility and staff for technical support and Coordination for the Improvement of Higher Education Personnel (CAPES).

This work was supported by FAPESP (São Paulo Research Foundation - Financial Support: 2014/50844-3).

All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee (nº 6611181016) from School of Veterinary Medicine and Animal Science of São Paulo University (FMVZ-USP, São Paulo, SP, Brazil).

The authors declare no conflict of interest.

Gustavo Sá Schiavo Matias and Paula Fratini conceived and designed the research. Gustavo Sá Schiavo Matias, Vitória Frias Batista and Paula Fratini conducted the experiments. Gustavo Sá Schiavo Matias, Ana Claudia Oliveira Carreira, Maria Angelica Miglino and Paula Fratini analyzed the data. Gustavo Sá Schiavo Matias and Paula Fratini wrote the manuscript. Maria Angelica Miglino for funding. All authors read and approved the manuscript.