Introduction

Periodontal disease is an inflammatory disease of the periodontium which can leads in tooth loss. Currently, is treated by tooth extraction, gingival surgery, root planning and conditioning, application of growth and differentiation factors or filler materials and guided tissue regeneration. Tissue Engineering (TE) strategies using a combination of scaffolds and mesenchymal stem cells (MSCs) has paved the way for new therapies.

The aim of this study was to isolate and characterize canine adipose-derived stem cells (cASCs) along its proliferative process, analyzing the expression of typical MSCs markers, and differentiation capacity in osteogenic and chondrogenic lineages, assessing osteoblasts and chondroblast gene expression and extracellular matrix production. In addition, we studied cASCs behavior when cultured onto a previously developed biodegradable bi-layered scaffold for periodontal regeneration.

Materials and Methods

cASCs isolation and characterization:

Adipose tissue (visceral and subcutaneous) was harvested from adult dogs, according to the animal welfare Portuguese legislation. cASCs were isolated by an enzymatic method and expanded along 5 passages in basal medium. cASCs were cultured for up to 35 days using either osteogenic medium (supplemented with ascorbic acid, dexamethasone and b-glycerophosphate) or chondrogenic medium (with ascorbic acid, TGF-b1, ITS, L-proline and sodium pyruvate).

Real time RT-PCR analysis was used to quantify the relative expression of CD73, CD90 MSCs genes; COL1A1, OSTEOCALCIN and RUNX2 osteoblasts genes and COL2A1, COL10, COMP and SOX9 chondroblasts genes. Alizarin Red staining was performed to assess the presence of mineralized matrix, and Toluidine Blue, Safranin O and Alcian Blue staining to assess cartilage matrix deposition.

Scaffolds preparation:

Starch+poly(ε-caprolactone) fibre meshes were obtained by wet-spinning, using two different precipitation baths: methanol as a control (SPCL), and calcium silicate solution to allow the SPCL functionalization with silanol groups (SPCL-CaSi), which are osteoconductive.

Cell seeding/culturing onto the scaffolds:

Each material was seeded with 1×10⁵ cells and cultured in basal and osteogenic medium for up to 28 days: Cellular proliferation was characterized by scanning electron microscopy (SEM) and by double strand DNA quantification. Cell viability was studied by MTS quantification. In order to evaluate osteogenic differentiation, ALP and calcium quantification assays were performed, as well as Alizarin Red staining. In addition, real time PCR analysis was performed to quantify the relative expression of stem cells and osteoblasts typical markers.

Results and Discussion

PCR results revealed a decrease in the expression levels of CD73, CD90 in cASCs cultured in basal medium along passages. Relative expression of SOX9 and COL2A1 was higher in samples from visceral origin. Furthermore, it was observed an increase of expression of COL1A1 and RUNX2 genes in cASCs cultured under osteogenic stimulus. This expression seems to be higher in the samples from lower passages. Alizarin Red staining corroborates this data, showing an increase of mineralizated matrix along time, particularly in samples from lower passages.

Regarding cASCs performance onto the materials, SEM revealed cellular proliferation, both in basal and in osteogenic conditions. A higher quantity of calcium phosphate crystals was observed in SPCL-CaSi scaffolds as compared to SPCL in both culture mediums, confirming us the bioactivity of the first material. DNA increased along culture time, particularly in the functionalized scaffolds cultured in osteogenic medium. MTS metabolic product also showed increased values along time, particularly in SPCL-CaSi. ALP activity increased until the 21^th day and afterwards decreased. ALP was higher in SPCL, maybe due to a hypothetical negative effect of silicon under ALP activity.

Conclusions

The present work brought, in author’s knowledge, the first data about the osteogenic potential of cASCs along different culture periods.

Furthermore, this work showed cASCs in combination with this SPCL bioactive scaffold may be used in a TE approach to reach the regeneration of periodontal damages, namely, in its osseous component.