Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2

https://doi.org/10.1016/j.rvsc.2017.11.010Get rights and content

Highlights

  • Osteogenic differentiation can be stimulated using 5 and 7.5 mM CaCl2.

  • This was shown by nodule formation, ARS semiquantification and OP gene expression.

  • ASC proliferation was enhanced by 5 and 7.5 mM CaCl2.

Abstract

Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl2 in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and β-glycerol phosphate; (2) CaCl2 based protocol composed of 3, 5 and 7.5 mM CaCl2. Differentiation and proliferation were evaluated at 7, 10, 14 and 21 days post-differentiation induction using the alizarin red staining (ARS) detecting matrix calcification. Semi-quantification of cell protein content, ARS and alkaline phosphatase activity (ALP) were performed using an ELISA reader. Quantification of the transcription level for the common osteogenic markers alkaline phosphatase (ALP) and Osteopontin (OP) was performed using RT-qPCR. In the presence of CaCl2, a concentration dependent effect on the osteogenic differentiation capacity was evident by the ARS evaluation and OP gene expression. We provide evidence that 5 and 7 mM CaCl2 enhance the osteogenic differentiation compared to the OM protocol. Although, there was a clear commitment of ASCs to the osteogenic fate in the presence of 5 and 7 mM CaCl2, cell proliferation was increased compared to OM. We report that an optimized CaCl2 protocol reliably influences ASCs osteogenesis while conserving the proliferation capacity. Thus, using these protocols provide a platform for using ASCs as a cell source in bone tissue engineering.

Introduction

Musculoskeletal injuries are common causes of death or retirement among race horses (Johnson et al., 1994). In this context, limb fractures account for the majority of fatal injuries. Insufficient fracture healing can lead to bone nonunion and dysfunction with an unsatisfactory prognosis and complications such as delay in healing, fixation failure, and stress-induced laminitis (López Echenique et al., 2012). Among other techniques, fractures in horses are currently treated with external fixation by means of a cast and internal fixation by use of metal plates and screws. The efficiency of the treatment strategy could be improved via enhancing the healing ability of bone. Recently, bone regeneration methods relying on the use of mesenchymal stem cells (MSCs) in conjunction with tissue engineering have been proposed to shorten the process of fracture healing. This in part was due to the differentiation potential of MSCs into osteoblasts but also due to their immunoregulatory and paracrine effects (Uccelli et al., 2008, Berg et al., 2009, Arnhold and Wenisch, 2015). Nevertheless, bone healing is not only complicated in humans but also a major problem in veterinary medicine (Adamiak and Aleksiewicz, 2006, Welch et al., 1997). Therefore, the osteogenic differentiation potential of MSCs is worthwhile having closer attention.

The harvest of fat tissue is much easier and feasible in equine patients in comparison to obtain bone marrow for the derivation of MSCs, the number of studies using adipose tissue derived mesenchymal stem cells (ASCs) in veterinary medicine is continuously increasing. Although there are many reports describe the osteogenic differentiation potential of ASCs (Reich et al., 2012, Takemitsu et al., 2012, Vieira et al., 2012) compared to bone marrow derived MSCs, ASCs always seem to be the second choice. Bone marrow derived MSCs are reported to have a greater efficiency towards osteogenesis compared to ASCs. In that term, It was found that ASCs show a limited osteogenic capacity (Liu et al., 2007) and display a lower mineralization potential compared to bone marrow MSCs (Shafiee et al., 2011). However, the easy accessibility and the good proliferation potential of ASCs offer a great opportunity as an abundant cell source (Han et al., 2014). The standard osteogenic induction medium consisted of the basal medium supplemented by dexamethasone, β-glycerophosphate and ascorbic acid (Qu et al., 2007). Other osteogenic inducers for ASCs have also been described using mechanical stimulation by low dose ultrasound (Yue et al., 2013), overexpression of osterix; a zinc finger transcription factor (Wu et al., 2007), electroporation of Runx2 and osterix (Lee et al., 2011). On the other hand, combined Ca+ 2 (9 mM) and P ions (4.5 mM) induced proliferation, mineralization and osteogenic differentiation in the human periodontal ligament cells, while higher concentrations caused significant cell death (Lee et al., 2011). Therefore, to improve the osteogenic differentiation potential of equine ASCs not only for basic research but also for a possible therapeutic intervention, some factors need to be considered and discussed e.g. minimizing and optimizing the concentrations of osteogenic inducing factors. In this study, we hypothesized that optimized concentrations of CaCl2 supplement enhances the osteogenic differentiation capacity of ASCs. Therefore, we have tested a protocol using 3, 5 and 7.5 mM CaCl2 concentrations added to the basal medium compared to a standard osteogenic differentiation protocol including dexamethasone, glycerophosphate and ascorbic acid. The osteogenic differentiation potential was examined initially via morphological observation of cellular aggregation-like nodule and evaluation of the alizarin red staining (ARS) as evidence for calcium depositions. Additionally, semi-quantification for ARS and the alkaline phosphatase (ALP) activity was performed via measurement of the optical density using a microplate reader. Furthermore, quantification of the cell protein as an indicator for cell number was examined using the sulforhodamine B (SRB) assay. Also, quantification of the alkaline phosphatase (ALP) and osteopontin (OP) genes expression were measured using RT-qPCR. The data were collected at days 7, 10, 14 and 21 post-differentiation induction and were statistically analyzed using a two way analysis of variance (ANOVA).

Section snippets

Isolation and culture of ASCs

Subcutaneous adipose tissue was harvested from the region above the M. gluteus superficialis from 3 mixed breed horses (2 males and 1 female; aged mean ± SD, 4.75 ± 1.71 years) as previously described by Raabe et al. (2010). All samples were obtained from horses being slaughtered at the local abattoir. Briefly, adipose tissue was washed three times in phosphate-buffered saline (PBS, PAA, Germany) and was diced into small pieces and digested for 40–60 min at 37 °C with 0.1% collagenase type I (Biochrom

Evaluation of osteogenic differentiation

After the induction of osteogenic differentiation, ASCs commitment was initially evaluated via microscopic detection of nodule formation. At day 10 post-differentiation induction, the cellular aggregation was detected in the presence of OM, 5 and 7.5 mM CaCl2 medium compared to cells cultured in the BM. The nodule formation, is a common term given to the cellular clusters indicative for the osteogenic commitment (Fig. 1a and b). Next, we evaluated whether an evidence for the nodule calcification

Discussion

Cell-based therapies using MSCs have been reported frequently in equine medicine with an attempt to improve the limited intrinsic capacity for completion of self-repair of cartilage, tendon and bone after injury (Smith et al., 2003, Berg et al., 2009). The tri-lineage differentiation of adipose tissue derived mesenchymal stem cells and thus a possible therapeutic potential has been shown in many studies using standard differentiation protocols. A major issue to be solved by bone tissue

Competing interest

All the authors have declared no conflict of interest regarding the publication of this paper

Acknowledgement

The authors wish to thank Kathrin Wolf-Hofmann for expert technical assistance and Eva Kammer for the figures preparation. The authors wish to thank Kathrin Wolf-Hofmann for expert technical assistance and Eva Kammer for editing the figures.

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