Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells
Introduction
Numerous studies have shown that an organized extracellular matrix (ECM) promotes cell adhesion, differentiation and proliferation (1,2). The study of Bissell and Radisky (3) helped to establish the essential role of ECM in the maintenance of tissue homeostasis, and today it is well known that cells may enter apoptosis by an inappropriate interaction between them and the ECM, or because of a lack of communication and interaction between them. Apoptosis in response to an inappropriate interaction of cells with the ECM is known as anoikis, a type of cell death initiated by signals that are different to those that initiate other types of apoptosis (4). Anoikis has been described in various cell types, including epithelial cells (5), fibroblasts (6), osteoblasts (7) and neurons (8). This kind of apoptosis assumes great importance in studies of cell therapy, in which the therapeutic effectiveness is largely dependent on the survival of transplanted cells (9., 10., 11.).
In strategies of cell therapy for spinal cord repair, adult stem cells are usually injected into an injured tissue in which vascularization is low, and where a significant inflammatory response may be present. Moreover, in most cases there is a post-traumatic cavity and therefore, absence of ECM. In these adverse circumstances the transplanted cells may die by anoikis, and various studies have shown that the number of living stem cells drops significantly after transplantation in models of spinal cord injury (SCI) (12). For this reason, numerous studies have been carried out in order to obtain an ECM that allows the primary culture of stem cells, being able to inhibit, at least in part, the phenomenon of anoikis as a result of achieving a permissive environment for cell survival, proliferation and differentiation (13., 14., 15.). On the other hand, several authors have shown that the addition of trophic factors [brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF) and epidermal growth factor (EGF)] to culture medium and biologic matrices could protect the cells from apoptotic damage (9,16) and may be useful for inducing cell differentiation (17).
One of the scaffolds more widely used as ECM is collagen. It is one of the major components of ECM, and it seems to be able to induce axonal regeneration in some models of SCI (18,19). Other scaffolds with the ability to facilitate axonal elongation are alginate hydrogel (20,21), poly-alfa-hydroxy acid (22,23), synthetic hydrogels (24) and polyethylene glycol (25).
Matrigel is an ECM derived from Engelbreth Holm Swarm sarcoma containing laminin, fibronectin and proteoglycans, and diverse studies have shown its usefulness, inducing the proliferation of nerve cells in vitro and axonal regeneration, in diverse models of SCI (26., 27., 28.). Fibronectin is a glycoprotein found in the ECM and plasma that has also been used to form nerve guides (29). It participates in the migration and interaction with cells through their surface receptors.
One possibility for obtaining a biocompatible scaffold is to synthesize it from proteins such as fibrin, keeping in mind that the mesh that forms the protein must be sufficiently porous to allow cell migration and contact between cells and be able to integrate into the host tissue (30). Fibrin is the major component of a blood clot. It acts as a binding molecule for the interactions between cells. In injured tissue, some cells bind to fibrin through its surface receptors, which helps them to perform different biologic roles (31). Diverse authors have demonstrated the utility of fibrin in tissue regeneration (32), occasionally being used with a growth factor (fibroblast growth factor (aFGF) and neurotrophin 3 (NT-3)) or in combination with other matrices (33,34).
On the other hand, numerous in vitro studies have been conducted to assess the patterns of adhesion, migration capacity, morphology and viability of different cell types when they are seeded on fibrin gel (FG) (34). FG is used in surgical procedures as a biologic sealant, and considered to be a porous scaffold that can stimulate cell adhesion and growth (35). It is composed of two components: fibrinogen, a soluble glycoprotein of 340 kDa that is found in high concentrations in plasma (2–4 mg/mL), and thrombin, an enzyme needed for fibrin clot formation. Moreover, there is the possibility of developing autologous fibrin gel from a patient's own blood and enriching it with growth factors from platelets.
In a similar way, we can produce platelet-rich plasma (PRP) scaffolds. PRP was used successfully in 1970 by Matras for applications such as skin grafts on mice (36); since then numerous studies have shown beneficial effects on the repair of skin ulcers. At present, PRP is the autologous scaffold most often used in orthopedic (37) and maxillofacial surgery (38). It is obtained from blood by separation techniques and a subsequent gel can be obtained using a mixture of calcium chloride with thrombin. Diverse authors have described different methods for obtaining a platelet concentrate, introducing variations in the time of centrifugation and number of spins (39,40). The gel obtained is a concentration of platelets suspended in a small volume of plasma that acts as a carrier of growth factors, which are secreted actively by the platelets (41., 42., 43.). Furthermore, PRP is rich in proteins, such as fibronectin, as well as fibrin and adhesive proteins, that act as adhesion molecules and can promote cell migration. The main advantage is being an autologous material, without toxicity or an immune response. Given the growing emergence of cell therapy strategies for SCI (11,44), the aim of this study was to compare PRP and FG, with the purpose of knowing which of these two biologic matrices used as scaffolds provides the best conditions for increasing the viability and biologic activity of human bone marrow stromal cells (hBMSC) and promoting neural differentiation.
Section snippets
Isolation and characterization of hBMSC
Bone marrow (BM) samples were harvested from the trabecular bone of hematologically normal adult patients (aged from 52 to 60 years) undergoing routine total hip replacement surgery. Consent was obtained in all cases. A single BM aspirate (35–50 mL) was taken from each patient's iliac crest. To prevent clotting, the aspiration needle was primed with sterile heparin sulfate (1000 U/mL). The BM aspirate was aliquoted immediately after collection. Each 4 mL of the aspirate was layered onto 3 mL
Cell morphology after 7 days in culture
In the present study, hBMSC showed the following profile after phenotypic flow cytometry studies: CD29+, CD44+, CD105+, CD166+, CD34− and CD45−. FG scaffolds were consistent and fairly manageable, but hBMSC were not properly adhered to the fibrin mesh. A few hours after starting culture, cells showed a rounded morphology and formed groups. Only occasional cells with a typical mesenchymal morphology could be seen (Table III).
A liquid consistency was seen in 5/2 and 10/4 FG scaffolds. They were
Discussion
Numerous studies on cell therapy have been applied to various diseases (46., 47., 48.). Although the results confirm the ability of stem cells to repair damaged tissues, regeneration is limited by poor survival of transplanted cells. Most of the stem cells die in the first days after transplantation because of the adverse conditions at the site of administration (10,12). To increase the survival of these cells it is necessary to conduct a combined therapy using stem cells and biologic matrices
Acknowledgments
This work was supported by grants from the Carlos III Institute (FIS 07/0621, FIS PS09/01105) and Mutua Madrileña Foundation.
Declaration of interest: The authors report no competing financial interests exist. The authors alone are responsible for the content and writing of the paper.
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These authors contributed equally to this work.