Full length articleHarnessing Wharton’s jelly stem cell differentiation into bone-like nodule on calcium phosphate substrate without osteoinductive factors
Graphical abstract
Bone regenerative medicine.
Introduction
Bone tissue regeneration remains a significant and challenging endeavour in the field of orthopaedic and craniofacial surgery. Bone has a remarkable ability for healing and represents the only connective tissue that retains the ability to enlist all of the key prenatal signalling events during healing. However it cannot heal by regeneration if an injury is beyond a critical limit. Despite progresses in clinical treatments in recent years, several drawbacks exist such as risk of donor site morbidity for autografts harvest, pathogen transmission and rejection by the recipient’s body in case of either allografts and xenografts use, and poor osteoinductive and mechanical features of synthetic ceramics. Bone morphogenetic proteins (BMPs) have been employed in many preclinical and clinical studies exploring their osteoinductive potential in several animal models and human diseases. The initial excitement arising from the excellent clinical results of bone morphogenetic protein-2 (BMP-2) has been tempered significantly by increasing safety and cost concerns [1].
To promote in vivo bone formation, bone tissue engineering approaches involve the combination of stem cells (SCs) and bioactive materials [2]. Adult SCs are involved in tissue regeneration throughout a person’s life. Major bottlenecks in the use of adult SCs are their highly invasive harvesting procedure, low frequency and age-declining proliferation and differentiation potential. Therefore, the search for alternative sources for adult SCs is of significant value. So far, increasing success has been reported in the literature for isolation and characterization of stem cells from perinatal tissues as Wharton’s jelly [3], [4], [5]. Wharton’s jelly stem cells (WJ-SCs) are thought to constitute powerful candidates for regenerative medicine and driving them into the osteoblastic lineage represents a great promise for regenerating bone.
Desired bioactive materials would promote SC differentiation without any additional growth factors [6], [7]. Often, biochemists induce SCs differentiation by adding chemical factors in vitro such as dexamethasone or β-glycerophosphate, which are not encountered in the body. Recent innovative discoveries suggest the importance of intrinsic mechanical (elastic modulus) and biophysical properties of substrate (topography), in eliciting cellular effects compared to the traditional induction method using medium supplements that are not physiologically relevant [8], [9]. Among the numerous attempts to develop bone tissue interactive substrates, calcium phosphate (CaP) ceramics, obtained by either wet-chemical deposition (sol-gel, biomimetics…) or physical deposition (plasma spray), have received wide attention due to their similarities with the inorganic mineral phase of bone [10], [11]. However, limitations regarding harsh fabrication conditions, long-term stability and biocompatibility, and the requirement of expensive instruments, with respect to these coating techniques still exist. Moreover, the poor osteoinductive properties of these materials can lead to failure of the stem cell osteoblastic commitment [7].
On the basis of the bone structure, it is assumed that optimal osteoinductive coatings should possess several types of features as a rough surface, high stiffness strength close to that of the bone. Recently, a new and straightforward method based on spray-assisted deposition was used to design a bone inspired substrate [12]. This versatile method allows the construction of submicron-sized coatings with possible modulation of thickness and surface topography. The ease of adapting this spray technology in industrial production presents an additional advantage. Our hypothesis is that the intrinsic mechanical and biophysical properties of sprayed CaP are able to induce osteogenic differentiation and could be used as a substrate for bone tissue engineering. Here, we examine the effect of CaP substrate on WJ-SCs behaviour through cell mechanobiology and integrin expression. To our knowledge, for the first time, we demonstrate the in vitro sequences of WJ-SCs proliferation, differentiation and mineralization associated with three-dimensional bone-like nodule formation without any biochemical supplements.
Section snippets
Material
Calcium nitrate (Ca(NO3)2,4H2O), diammonium hydrogen phosphate ((NH4)2·HPO4) and Tris(hydroxymethyl) aminomethane (Tris) from Sigma were used without further purification. The salt solutions were prepared in ultrapure water (Millipore®). Calcium solution of Ca(NO3)2·4H2O (0.32 M) and a phosphate solution of (NH4)2 HPO4·(0.2 M) were prepared in Tris buffer (10 mM Tris, pH 4 and pH 10 respectively). Coverslips glass of 14 mm diameter were provided from Thermo Scientific. Each experiment was preceded
Results and discussion
The CaP substrates were prepared at room temperature by simultaneous spraying of Ca(NO)3, 4H2O and (NH4)2HPO4 aqueous solutions on a glass coverslip for 2 s followed by a 2 s spraying of the rinsing solution followed by 2 s of drying. This was repeated 50, 100 and 150 times to get three coated surfaces (CaP50, CaP100 or CaP150). Chemical and physical properties of these coatings, including homogeneity, calcium phosphate phase and their bioactivity, elastic modulus and surface topography,
Declaration of interest
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare that they have no competing interests.
Acknowledgments
The authors are very grateful to the staff of Reims Maternity Hospital for providing umbilical cords and the staff of the Core URCACyt and PICT (URCA). This work was partially supported by IMACELL from CNRS project, France. S. Mechiche Alami was supported by a doctoral fellowship of la ville de Reims. We thank E. Mathieu from INSERM U1121 (Université de Strasbourg, France) for technical help concerning transmission electron microscopy, N. Bouland for technical help concerning histology, Dr. S.
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Both authors have contributed equally to this work.