Bone Tissue Engineering: Scaffold Design and In Vitro Testing Report

This report delves into the field of bone tissue engineering, specifically addressing the clinical need for improved bone graft alternatives. It examines the current treatment regimes, quantifying the impact of bone fractures and defects, and highlighting the need for more accessible and effective solutions. The report then focuses on scaffold design, exploring essential features such as biocompatibility, biodegradability, porosity, and mechanical properties. It details the selection of polymers, with a focus on chitosan, and discusses the importance of scaffold architecture in promoting cell adhesion, nutrient transport, and waste removal. Furthermore, the report outlines in vitro testing methods, including cell line preparation, experimental design, and cell proliferation and viability assays, to assess the immunogenicity and effectiveness of the scaffolds. The report concludes by emphasizing the potential of tissue engineering to revolutionize bone repair and the need for continued research to develop more efficient and cost-effective treatments.

Biomedical Engineering 1
BIOMEDICAL ENGINEERING
By [Name]
Course
Professor’s Name
Institution
Location of Institution
Date

Biomedical Engineering 2
Biomedical Engineering
Introduction
Tissue engineering is the latest technology that focuses on the
restoration of the functions of an organ or tissue after diseases, injury or due
to aging. Bones fracture as a result of accidents, ageing, degenerative
diseases, and wars. Treating bone defects using tissue engineering is the
best available clinical approach since the patient’s own tissues may
ultimately get involved in the repair. In recent days, well-developed methods
in tissue engineering have been adopted to enhance bone transplanting.
Studies on the scaffold are used to repair bones and bone regeneration by
providing a scaffold which acts as an artificial extracellular matrix. It is also
used in the engineering of cartilage.
Bone transplants
There is high demand for bone grafts with the U.S. having over half a
million of its population undergoing bone grafting annually. Globally, about
2.2 million people undergo bone grafting each year (Villa et al. 2015, 251).
These grafts costs more than $2.5 billion dollars annually (Qasim, Chae and Lee
2019, p.4333). These numbers are expected to double by 2020 and get to
$11.5 billion by 2025 owing to the increasing demands of this generation
(Villa et al. 2015, 251). Clearly, there is a need to adopt more affordable and

Biomedical Engineering 3
accessible treatment alternatives to rectify defective bone tissues, and, thus,
this paper comes handy to do explain how.
Features of Scaffolds
Compatibility
Biocompatibility is the ability to be compatible with body organs. The
mechanical properties of the scaffold should be similar to those of the tissue
of the original bone. Different materials can be used to make scaffolds for
bone regeneration. These include polymers or the mixture of polymers with
calcium phosphate materials such as hydroxyapatite, and other substances
which include single-walled or multi-walled carbon nanotubes (Tangsadthakun et
al., 2017). Cells must be able to function normally, adhere, and migrate onto
the surface and eventually through the scaffold into the matrix (Villa et al.
2015, 251). Besides, the implanted tissue must demonstrate a high degree of
negligible immune reaction after implantation. This prevents allergic
responses, which might hinder the healing process or even face rejection by
the body.
Sterility and pyrogenicity

Biomedical Engineering 4
This is a characteristic feature of injectable products. This is because
scaffolds are made with the intention of being implanted into the body of a
human being. In other words, the mechanical properties of scaffolds should
be consistent with the body's anatomy (Ghassemi et al. 2018, p.90). In engineering
bone cartilages, producing scaffolds with compatible mechanical
characteristics has become one of the significant challenges (Ghasemi-
Mobarakeh 2015, p.728). Another problem concerns different healing rates
because of age differences. For instance, the rate of healing in children is
faster, about six weeks, unlike in adults, where it can even take ten weeks
(Tangsadthakun et al., 2017; Qasim, Chae and Lee 2019, p.4333). These realities,
however, have opened up an area of interest. The current technologies
provide scaffolds with higher porosity and vascularization.
Biodegradability
The scaffold material should be degradable for easy and safe
replacement of the material used to make the scaffold with osteoid deposits.
The main focus of tissue engineering is to give time to the cells within the
body to eventually replace the implanted tissue or scaffold (Zhang et al. 2016, p.
669; (Ghassemi et al. 2018, p.90). For this reason, therefore, the scaffold should be
biodegradable to allow cells to make their own extracellular matrix (Ghasemi-
Mobarakeh 2015, p.728; (Mirhadi, Nourbakhsh, Lotfian and Hosseini 2015, p.1363). Besides,
the products of biodegradation should not have toxic substances to allow
them to be easily removed from the body without interfering with other

Biomedical Engineering 5
organs (O'brien 2011, p.90). The only way to enable degradation to co-occur with
tissue formation is the application of inflammatory responses coupled with
the controlled infusion of cells like the macrophages.
Porosity
The architecture of the scaffold is instrumental and should be
considered. The scaffold should be porous to allow the entry of nutrients,
penetration of cells, and transfer of water. Also, the porous structure allows
the passage of cellular wastes and products of scaffold degradation to freely
exit without any difficulty (Ghasemi-Mobarakeh 2015, p.728). Of interest also, is the
small size of pores. Scaffold which has been synthesized from natural
materials like collagen have ligands that provide cites through which cells
interact with scaffolds. It follows that the density of ligands is influenced by
surface area, which depends on the size of the pores (O'brien 2011, p.90).
Therefore, it is essential to have large pores to allow the migration of cells
into the scaffold and facilitate the binding of cells to the scaffold. However,
to facilitate efficient coupling, a fixed size of pores is critical, although this
varies depending on the tissue used.
Viability
The manufacturing technology of the scaffold is also important to put
into consideration. For a scaffold to become viable both commercially and
clinically, it is crucial to keep it cost-effective and possible to produce in

Biomedical Engineering 6
small quantities in the laboratory (Ghasemi-Mobarakeh 2015, p.728). Transferring
tissue engineering to clinical practice depends on the development of
scalable manufacturing processes. Important to note also is the mode of
delivery of the scaffold which determines its storage.
Physical characteristics
Furthermore, scaffolds should possess all the physical attributes to
allow them to bear the weight of the body without breaking during
amelioration. Due to its strength, the bone tissue has an anisotropic
characteristic since its power is dependent on the way the load imposed on it
is oriented. It also depends on the resistance to high pressure (Ghasemi-
Mobarakeh 2015, p.728). The strength of the bone tissue depends on its size and
position in the body. Therefore, because of the reasons above, a definite
structure, composition, and shape of the scaffold are necessary to restore
the needs of the bone.
Furthermore, there should be a balance between the various variables within
the bone to find the desirable characteristics that are compatible with the
damaged tissue. Lastly, the scaffold should have a controlled delivery of
drugs and bioactive molecules. The application of scaffolds in the clinical
field shows a better ability to facilitate to repair bone.
Scaffolds

Biomedical Engineering 7
One of the best polymers to use in making 3D scaffolds is the natural
polymers like chitosan since they are flexible and can be modified into any
shape. Chitosan is a first polymer in creating 3D scaffolds because it stands
out among other polymers (Villa et al. 2015, 251). Chitosan has several factors
that enhance its capacity to stand out among other polymers. Among these
characteristics is the ability of chitosan to open the tight junctions which
exist between the epithelial cells in the epithelial (Sainitya et al. 2015, p.485). In
conjunction with mucoadhesion, this property enhances the permeability of
the trans-mucosal region and also improves the transportation of substances
like drugs (Dorati et al. 2017, p.96).
Furthermore, chitosan is soluble in acidic pHs and water but insoluble
in neutral solutions, a property which is essential for its functioning in the
body (Sainitya et al. 2015, p.485). Besides, chitosan is hydrophilic and has a
positive charge, a property that allows it to freely interact with harmful
polymers and other polyanions in aqueous solutions (Dorati et al. 2017, p.96).
Also, the positive charges of chitosan are essential in improving adhesion to
the human mucosal surface. As a result of this property, chitosan has widely
been used in the delivery of drugs in the mucosal region. Apart from
chitosan, other substances such as polypropylene and
polymethylmethacrylate are the polymers used to make scaffolds.
Treatment options

Biomedical Engineering 8
Several treatment options are available in case of scaffolding. One of
the most popular treatment options is the use of bioactive molecules.
Bioactive molecules enhance the viability and survival of grafts hence
promotes angiogenesis (Gomes et al. 2015, 349). Bioactive chemicals like drugs
and growth factors can be incorporated into scaffolds. Those biomolecules
which can quickly be released and interact with cells can be controlled by
releasing them by diffusion (Ghasemi-Mobarakeh 2015, p.728). These cells produce
more growth factors which stimulate cell growth. Biomolecules can exist in
two forms: short peptide sequences and whole protein molecules (Villa et al.
2015, 245). The most preferred ones for use are the short peptide molecules
since whole protein molecules fold, reducing the availability of the binding
proteins.
In vitro Assays
In vitro tests are used to enhance the understanding of the
immunogenicity of scaffolds. Primarily, it is the tenocytes which are assessed
using live dead staining (Knight, E. and Przyborski 2015, p.750). This means that
while phenotypic retention is checked through real-time PCR analysis of
tenocytic genes, in vitro tests are mainly extracellular. Skill-like material is
used (Achatz et al. 2016, p.276).
Cell line

Biomedical Engineering 9
An In vitro test involves the preparation of the cell line. Cellular assays
are performed using the C2C12 cell line (Gomes et al 2015, p.358). The analysis is
performed by continually passing the cell line on flasks with Eagle's medium,
which has high glucose content and 10% fetal bovine serum. It also has
antibiotics (Tangsadthakun et al. 2017). The mixture is maintained at 37oC and 5%
CO2.
Experimental design
The test begins by placing a scaffold with a diameter of 4.5 mm and a height
of 2.5 mm into a 48-well plate. After that, seeded with 10 μL of the entire medium
whose contents are 2x105 trypsinized cells per scaffold drop wise onto them (Gomes et al. 2015,
p.350). This is followed by the incubation of the materials at 37oC in 5% of CO2 atm for a total
of 45 minutes to enhance the attachment of cells on the scaffold (Knight and Przyborski, 2015,
p.749). To minimize any errors resulting from contamination before any measurements are
taken, each scaffold is transferred to a new well. Afterward, a new culture is added. The cultures
are performed in three points that is, after 24, 72, and 140 hours.
Cell proliferation and viability assay
The proliferation of cells at each point is tested using Invitrogen. The manufacturer’s
instructions are followed strictly. Invitrogen is added to a culture containing 40μl of a reagent in
a medium of 400 μl. The mixture is incubated for 37oC for 90 minutes, after which it is
transferred to new wells (Sainitya et al. 2015, 485). The mixture is passed through a fluorescent
emission at 590nm and the readings recorded. The viability of the cells is also tested using
calcein-AM assay.

Biomedical Engineering 10
Conclusion
Tissue surgery is one of the latest technologies that have improved lives drastically. In
case of an injury, one is assured that their body parts will be intact. However, it is difficult to
deny that the process in overly expensive as we have seen. Cheaper alternatives can, therefore,
be explored to ensure this is accessible by everyone.

Biomedical Engineering 11
References
Achatz, F., Kujat, R., Pfeifer, C., Koch, M., Nerlich, M., Angele, P. and Zellner, J., 2016. In vitro
testing of scaffolds for mesenchymal stem cell-based meniscus tissue engineering—introducing a
new biocompatibility scoring system. Materials, 9(4), p.276.
Dorati, R., DeTrizio, A., Modena, T., Conti, B., Benazzo, F., Gastaldi, G. and Genta, I., 2017.
Biodegradable scaffolds for bone regeneration combined with drug-delivery systems in
osteomyelitis therapy. Pharmaceuticals, 10(4), p.96.
Ghassemi, T., Shahroodi, A., Ebrahimzadeh, M.H., Mousavian, A., Movaffagh, J. and Moradi,
A., 2018. Current concepts in scaffolding for bone tissue engineering. Archives of Bone and Joint
Surgery, 6(2), p.90.
Ghasemi-Mobarakeh, L., Prabhakaran, M.P., Tian, L., Shamirzaei-Jeshvaghani, E., Dehghani, L.
and Ramakrishna, S., 2015. Structural properties of scaffolds: crucial parameters towards stem
cells differentiation. World journal of stem cells, 7(4), p.728.
Gomes, S.R., Rodrigues, G., Martins, G.G., Roberto, M.A., Mafra, M., Henriques, C.M.R. and
Silva, J.C., 2015. In vitro and in vivo evaluation of electrospun nanofibers of PCL, chitosan and
gelatin: A comparative study. Materials Science and Engineering: C, 46, pp.348-358.
O'brien, F.J., 2011. Biomaterials & scaffolds for tissue engineering. Materials today, 14(3),
pp.88-95.
Knight, E. and Przyborski, S., 2015. Advances in 3D cell culture technologies enabling tissue‐
like structures to be created in vitro. Journal of anatomy, 227(6), pp.746-756.

Biomedical Engineering 12
Mirhadi, S.M., Nourbakhsh, A.A., Lotfian, N. and Hosseini, B., 2015. Strength development,
bioactivity and biodegradability of forsterite nanostructure scaffold. Ceramics
International, 41(1), pp.1361-1365.
Sainitya, R., Sriram, M., Kalyanaraman, V., Dhivya, S., Saravanan, S., Vairamani, M., Sastry,
T.P. and Selvamurugan, N., 2015. Scaffolds containing chitosan/carboxymethyl
cellulose/mesoporous wollastonite for bone tissue engineering. International journal of
biological macromolecules, 80, pp.481-488.
Tangsadthakun, C., Kanokpanont, S., Sanchavanakit, N., Banaprasert, T. and Damrongsakkul,
S., 2017. Properties of collagen/chitosan scaffolds for skin tissue engineering. Journal of Metals,
Materials and Minerals, 16(1).
Villa, M.M., Wang, L., Huang, J., Rowe, D.W. and Wei, M., 2015. Bone tissue engineering with
a collagen–hydroxyapatite scaffold and culture expanded bone marrow stromal cells. Journal of
Biomedical Materials Research Part B: Applied Biomaterials, 103(2), pp.243-253.
Zhang, B., Montgomery, M., Chamberlain, M.D., Ogawa, S., Korolj, A., Pahnke, A., Wells,
L.A., Massé, S., Kim, J., Reis, L. and Momen, A., 2016. Biodegradable scaffold with built-in
vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nature
materials, 15(6), p.669.
Qasim, M., Chae, D.S. and Lee, N.Y., 2019. Advancements and frontiers in nano-based 3D and
4D scaffolds for bone and cartilage tissue engineering. International journal of
nanomedicine, 14, p.4333.