In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells 1

PURPOSE: To evaluate in vitro ability the of three different biomaterials – purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane – as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC) intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.


Introduction
Stem cells are defined by their ability for self-renewal and survival while maintaining genomic integrity [1][2][3] . The bone marrow has at least three stem cell populations -hematopoietic stem cells, endothelial progenitor cells and also mesenchymal stem cells (MSCs) -which are precursors of non hematopoietic tissues [1][2][3][4] . The MSCs are able to differentiate themselves into other phenotypes including those that produce cartilage, muscle tissue, bone, medullar stroma, tendon/ligament, and other connective tissues when exposed to an appropriate stimulus 1,[5][6][7] . In vitro, the MSCs may be grown directly or after density gradient separation; however, cell density is a critical factor affecting the cell growth 8 .
Morphologically, the MSCs are mostly fusiform and cuboidal 7,9 . A standard protocol to isolate bone marrow MSCs is based on their expansion potential and on the adherence of marrow-derived fibroblast-like cells, and lack of adherence of marrow-derived hematopoietic cells, to the plastic substrate of the cell culture plate [8][9][10][11] . In addition, MSCs possess immunophenotype characteristics as well as specific cell-surface markers, and are negative for hematopoietic markers such as CD3, CD14, CD19, CD34, CD38 and CD66 but positive for CD105, CD166, CD54, CD 55, CD13 and CD44 6,7,9,11,12 . The characterization is generally accomplished on culture-expanded cells and not on primary cells 8 .
Since the repair of large bone defects still poses a challenge for the orthopaedic, reconstructive and maxillo-facial surgeon 13 , several approaches have been used to treat them [13][14][15][16][17] . Although the autogenous cancellous bone graft is the most effective treatment for inducing bone regeneration and repair since it provides viable osteogenic cells 18,19 , it has disadvantages such as the requirement for a second surgical site, insufficient sites, and morbidity at the donor site 16,19,20 . Thus, bone-graft substitutes including hydroxyapatite, tricalcium phosphate, coral-collagen composite, natural coral, calcium carbonate-based ceramics and collagen combinations have been developed to treat a number of orthopedic diseases [19][20][21][22] . In addition, some of these materials allow direct anchorage to the local tissue. Due to this characteristic, these biomaterials have been used as scaffolds in reconstructive surgeries with excellent results while avoiding the use of bone grafts 23 . However, it is recognized that some biomaterials lack either osteogenic or osteoinductive properties 19,24 .
According to tissue engineering concepts it is possible to regenerate several tissues or organs by using mature cells or stem cells seeded onto adequate three-dimensional scaffolds 9,19,24 .
Vats et al. 25 described the steps involved in the engineering of tissues and organs. These include cell harvest from the donor site, seeding of cells onto a scaffold, stimulation of cellular proliferation, maintaining or stimulating cellular specialization or differentiation and, finally, transplant of the living tissue or organ to the patient. The final goal is the creation of a stable, complex three-dimensional construction of clinically useful size in vivo, combining all these steps.
An ideal scaffold for bone tissue engineering must provide an appropriate environment for tissue development; it should favor cell attachment, growth and differentiation and have biocompatible components 19,26 . Scaffolds may be derived from biological materials such as extracelullar matrix, plants and algae; or synthetic materials including hydroxyapatite, tricalcium phosphate ceramics, polylactide and polyglycolide, or a combination of these 9,27 . However, the scaffolds that deliver MSCs should have the following characteristics: be mechanically sensitive to the implantation site, be able to osteointegrate into the host tissues or disappear, be porous, allow cell attachment, growth and differentiation, permit bioactive molecules to have access to cells, provide maximal bone growth through osteoinduction and/or osteoconduction, and be sterilizable without loss of properties 5,19 .
The differentiation capacity of cultured bone marrow-derived MSCs coupled with the apparent ease of ex vivo culture manipulation has engendered considerable interest in potential therapeutic applications of these cells in a wide range of settings 28 .
Furthermore, the anti-proliferative and anti-inflammatory effects shown by these cells have also provided a basis for their application to disease therapies 7 . Therefore, the association of these biomaterials and cells with osteogenic potential may be a viable and promising alternative in the treatment of bone defects as observed in animal models 2,9,19,24 . Thus, the aim of this study was to investigate three different types of commercially available biomaterials as biocompatible 3D scaffolds for canine MSCs, intending bone tissue engineering.

Methods
The present study was performed according to the guidelines for the care and use of laboratory animals and was approved by the Ethics Committee from the School of Veterinary Medicine and Animal Science, UNESP at Botucatu.

Cell seeding and morphology
The scaffolds were placed in a 75 cm 2 cell culture flask at the previously described concentration. MSCs were seeded onto the scaffolds at a density of 2x10 6

Cell differentiation markers
After seven days of culture, Alizarin Red staining revealed no calcium deposition in the cell monolayer for the BioOsteo and Bonefill (Figures 2a and 2b). However, the hydroxyapatite showed little deposition of extracellular calcium in the hydroxyapatite (Figure 2c). Anti-Sp7/Osterix antibody was positive only in relation to cells cultivated in the presence of hydroxyapatite granules (Figure 2d).    The biomaterial hydroxyapatite may be of natural origins (derived from corals or bovine bone) or synthetic (hydroxyapatite, unsintered apatite or calcium-deficient apatite) 37 . Synthetic hydroxyapatite may be prepared as a dense non-porous or porous form, in either blocks or granules 15 . In the present study, a granular synthetic hydroxyapatite produced by the precipitation method was utilized. The use of hydroxyapatite for tissue engineering has been described for many years, especially due to its osteoconductive property 38  This finding stands in contrast to a prior study in which 95%

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells
of the cells seeded in hydroxyapatite matrix had adhered to its surface when observed only one day after seeding 36

Conclusions
All tested biomaterials were able to support mesenchymal stem cell adhesion and proliferation, and may be used as scaffolds for MSCs in bone tissue engineering. BioOsteo enhances hydroxyapatite-like crystal deposition and hydroxyapatite promotes osteogenic differentiation in early evaluation (seven days).