Natural Scaffold, from Bovine Bone Marrow, Reproduces Native Microenvironment and Supports CD34+ and Stromal Cells
Authors: Rosa Renata Giardini , Bianco Juares E. Romero , dos Santos Gabriela Pereira , Waldman Stephen D. , Weber Joanna , Joazeiro Paulo P. , Saad Sara Teresinha Olalla 
Published: October 2019

Abstract
Background: The idea of studying bone marrow outside its native environment is attractive and ideal. Due to the many functions of extracellular matrix (ECM), currently there is an interest in creating an environment that mimics the ECM present in the tissue, similar to the microenvironment in vivo. Molds replacing the ECM (scaffolds) have a porous structure and may assist the tissue regeneration by forming a suitable environment for adhesion, migration, proliferation and cellular differentiation. The appropriate ECM is a key factor as ECM proteins are site-specific and provide protein 'footprints' of previous resident cells. Because ECM proteins are among the most conserved proteins, the removal of xenogenic/allogenic cellular contents via decellularization could theoretically produce an essentially minimally immunogenic scaffold with a native intact structure for new tissue regeneration. Thus, the search for a scaffold that could be used to assess the behavior of cells and their interactions with the ECM in vitro/in vivo, and has different niches in its composition is highly desirable.
Aims: In recent years, a large number of molecular and cytogenetic abnormalities have been identified in AML, MDS and multiple myeloma, many of these defects can serve as markers for diagnosis/prognosis or as therapeutic targets. However, there are still many unknown molecular factors involved in genetic abnormalities or signaling pathways that contribute to the pathogenesis of the disease. Another very important aspect of these diseases is that they all are related to the mutual interaction of neoplastic cells and the microenvironment of bone marrow. In the absence of an ideal model or even the difficulty in reproduce a native environment, we proposed the characterization of a natural scaffold, from bovine bone marrow, which can be used as a study model, previously patented by our laboratory.
Materials and Methods: Bone marrow was decellularized by one or more incubations in an enzymatic digestion solution and polar solvent extractions, comprising an extracellular matrix with well-preserved 3D structure. Scaffolds were analyzed after the decelularization process for potential changes in structure (TEM, SEM, Histological staining, and immunohistochemistry for collagen III, IV, fibronectin) and mechanical properties. To verify if the scaffold would hold and support cell survival and extracellular matrix production, an in vitro study was performed using CD34+ (non-stromal) and HS-5 (stromal) cells. Cell-seeded decellularized scaffolds were cultured for 7-14 days and analyzed for Histological staining.
Results: Histology sections (H&E staining), TEM and SEM demonstrated the structure and ultrastructure of the processed matrix and confirmed both cellular extraction and preservation of the macroscopic 3-D architecture of the collagen fibers, blood vessels, and preservation of an organized matrix. Also, the decellularized scaffold was quite comparable to the native tissue in terms of its mechanical properties. Immunohistochemistry of the scaffold showed that the main components of the ECM were preserved. The in vitro experiments of both stromal cells (HS-5) and non-stromal cells (CD34+) demonstrated that they were able to adhere and in the HS-5 case also produce ECM during 7-14 days of culture. In both cases, an increase in cell number was observed and CD34+ overtime formed cluster and with 14 days of culture the cluster formation increased in size.
Conclusions: The results demonstrated that the decellularization process was efficient in keeping a 3-D structure and mechanical properties with a well-organized-preserved ECM. In vitro experiments showed that both CD34+ and HS-5 were able to proliferate and adhere in specific sites of the scaffold, suggesting that they were able to recognize their native environment. HS-5 produced ECM indicating that the scaffold worked as an optimal microenvironment. In conclusion, the scaffold could be used as a model, which has the potential to mimic the native microenvironment to enable research/studies of factors that are involved in self-renewal and maintenance of neoplastic cells in bone marrow. Also, this model could be very useful for pharmacological testing of bone marrow in vitro.

Disclosures
No relevant conflicts of interest to declare.