Collagen scaffold for mesencyhmal stem cell from stromal vascular fraction (biocompatibility and attachment study): Experimental paper

Background One of the most important part of tissue engineering (TE) is a matrix called scaffold. A good scaffold integrates with the host tissue and support the growth and differentiation of the cells. Collagen is the most abundant protein in the ECM and has been considered to be a group of proteins with a characteristic molecular structure—fibrillar structure, which contributes to the extracellular scaffolding. Objective In this research we study the biocompatibility and attachment of collagen scaffold by measuring the level of availability of mesenchymal stem cell (MSC) cluster from stromal vascular fraction (SVF). Method This study was experimental invitro on MSC culture derived from SVF, with post-test control group design. Biocompatibility was measured by viability of MSC from SVF with marker Propidium Iodine through flowcytometry and electron microscope was used to assess the population density of MSC from SVF by measuring the number of cluster cells seen. Result Oxidize cellulose has the greatest value of MSC cluster with average number of 2003 cell cluster. This result was significant with p < 0.05 using One-Way Anova and Tukey Test. Conclusion Collagen scaffold is ideal for MSC from SVF because of its compatibility and attachment.


Introduction
Tissue engineering is a multidisciplinary science that applies the principles of bioengineering for the fabrication of new and improved biomaterials capable of maintaining and restoring the functionality of organs and tissues impaired by disease and trauma. This translational approach has been applied to develop and design patient-specific tissue grafts that mimic the functional properties of native tissues. Three important factors have been accredited to the success of tissue engineering: cocultured stem cells, signaling factor, and the bio-fabricated scaffold [1].
Fundamentally, the tissue engineering paradigm consists of scaffolds, signals, and cells, These 3 elements can be combined or used independently to attempt to generate tissues in a limitless number of arrangements. However, with an increasing complexity of design, there are greater challenges to translation. For example, receiving regulatory approval for an acellular scaffold requires substantially less time and fewer resources than does a drug-eluting scaffold that has been preseeded with stem cells [1].
The stem cells are capable of differentiating into several types of tissues and organs, while the bio-fabricated scaffold provides structural support to the seeded stem cells. Signaling factors are responsible for influencing cell phenotype, metabolism, migration, and organization. Stem cells are undifferentiated cells of embryonic, fetal origin, and they possess the ability to give rise to differentiated cells and then finally develop into organs. Stem cell characteristics include the ability to selfreplicate and renew, clonage forming, and high potency ability. In terms of the potency ability of stem cells, stem cells can be totipotent, could differentiate into any cell types (pluripotent), and could differentiate into cells that arise from the three germ layers-ectoderm, endoderm, and mesoderm-from which organs develop. Stem cells can be categorized broadly into embryonic and adult stem cells and are efficient cell sources for tissue regenerative applications. They have also been reported to have the abilities to promote tissue homeostasis, growth, and repair, thereby contributing importantly to tissue and organ regeneration. Bio-fabricated scaffolds consist of decellularized biomaterials to provide structural and anatomical functions to the seeded stem cells, thereby resulting into successful formation of specific tissue [2].
MSCs are stromal stem cells that are heterogeneous and are derived from several tissue sources that include adipose tissue, periodontal ligaments, bone marrow, umbilical, placenta, and lungs. MSCs express surface markers like CD73, CD44, CD90, and CD105. The most widely known and used MSCs are bone marrow MSCs and adipose tissuederived MSCs isolated and purified from the bone marrow and adipose tissue, respectively [3].
Stem cells have self-renewal and multipotency abilities that may help in creating advanced tissue engineered skin substitutes. Among the main sources of cells that may be used to engineer such substitutes are adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These cells have the potential to secret paracrine factors, making them an attractive option for the treatment of acute and chronic wounds. So far, adult stem cells, especially mesenchymal stem cells (MSCs), have been widely used for regenerative healing. MSCs can selfrenew and have the ability to differentiate into osteoblastic, adipocytic, and chondrocytic lineages [4].
Collagen is the most abundant protein in the ECM and has been considered to be a group of proteins with a characteristic molecular structure-fibrillar structure, which contributes to the extracellular scaffolding. That is to say, collagen plays an important role in maintaining the biological and structural integrity of ECM and provides physical support to tissues. Collagen possesses extensive sources (such as bone, cartilage, tendon, ligament, blood vessel, nerve, skin), as it is the main structural protein of most hard and soft tissues. In addition, collagen offers low immunogenicity, a porous structure, permeability, good biocompatibility and biodegradability and has functions to regulate the morphology, adhesion, migration and differentiation of cells. All of these good performances make this natural polymer seem to be a promising biomaterial for scaffolds in tissue engineering. However, the collagen scaffolds lack mechanical strength and structural stability upon hydration, which limit their applications in particular tissues. Intermolecular cross-linking of collagen scaffolds can be achieved by physical or chemical methods, which can improve the mechanical properties of the scaffold. Besides, blending collagen with other materials, such as natural, synthetic polymers and inorganic materials, is also frequently used to enhance the mechanical strength of collagen scaffolds. Meanwhile, biochemical factors could be added into or modified onto the scaffold selectively according to the damaged region to improve the cellular outcome [5].
In this study we want to know biocompatibility and attachment of collagen scaffold for mesenchymal stem cell derived from stromal vascular fraction.

Methods
This study was experimental invitro on MSC culture derived from SVF, with post-test control group design. This study was conducted at Biomedic Laboratory of Faculty of Medicine Universitas Brawijaya Malang.
Collagen scaffold was obtained from commercially use biomaterial with the brand Lyostipt Oxidize cellulose obtained from commercially use biomaterial with the brand surgicell Stromal Vascular Fraction was originated from Rattus novergicus fat tissue with ZUK method [6].
Biocompatibility was measured by viability of MSC from SVF with marker Propidium Iodine through flowcytometry.
Electron microscope: Assessment of population density of MSC from SVF by measuring the number of cluster cells seen in imaging scanning electron microscope/SEM to reduce measurement bias, then measured the number of cluster cells by two observers, namely researchers and expert analysts in the field of stem cells. In addition, cells measured for density on SEM imaging were performed under a light microscope to prove that cells imaged in SEM were adipocyte derived stem cells.

Result
From Table 1 and Table 2 we know that oxidize cellulose has viability only 19.528, collagen 93.273 and control 95.995. Since the data is not homogen we proceed to parametric analysis, kruskal wallis test and Mann Whitney test.

Kruskal wallis examination
Based on the results of the Kruskal Wallis analysis, found that the p value for the Cell Number is 0.003. Because for the parameter Cell Number has a value of p < 0.05, which means that there is a significant difference in effect between treatments at an error rate of 5%

Mann whitney examination
Mann Whitney test results in Table 3 showed that the control group gave significant differences with all treatment groups. Likewise, the treatment group for Oxidized Cellulose has a significant difference with all treatments and controls.
These are an electron microscope examination of the MSC attachment to scaffold showed in Fig. 1.
Electrone microscope Scanning from mesenchymal stem cell to oxidize cellulose with 3000x magnification.
Since the data homogen and normally distributed, we used parametric study, One way Annova and Tukey Test.

One way Annova Examination
Based on the ANOVA analysis results it is found that the p value is 0,000 which means that there is a significant difference in effect between treatments at an error rate of 5%.
Tukey's test results in Table 5 showed that the control group with the oxidize cellulose group, and collagen had a significant difference. The

Discussion
MSC cultures derived from SVF combined with scaffold and then tested for viability. The results of the study showed that oxidized cellulose had very low viability at only 19.5%. This is consistent with research from (Yen et al.) where from the study cell viability combined with oxidized cellulose was 0%. There are certain materials present in the oxidized cellulose that are toxic to cells. Collagen has a better viability test with 93.237 almost the same with the control group. It show that collagen much more better for biocompatibility compare to oxidize cellulose [7].
From the electron microscope we can see the attachment of MSC to the scaffold. Both of the scaffold has the ability to attach with MSC. Although oxidize cellulose has a better result, but in viability test oxidize cellulose has a very low degree of viability, so we choose the collagen for the scaffold of MSC from SVF because of its compatibility and attachment.