This document discusses approaches for developing gradient biomaterials suitable for interface tissue engineering. It explains that gradient biomaterials can regulate cell behavior during tissue formation at interfaces between soft and hard tissues. The document then describes various types of gradient biomaterials, including those involving material composition, structure, physical properties, and biological factors. It also outlines several micro and nanoengineering techniques that can be used to fabricate gradient biomaterials, such as salt leaching, gas foaming, photolithography, and electrospinning.

1. MICRO AND NANO ENGINEERING
APPROACHES TO DEVELOPING GRADIENT
BIOMATERIALS SUITABLE FOR INTERFACE
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2. INTRODUCTION
■ Interface tissue found between soft and hard tissue regions such as
cartilage-bone, tendon-bone, ligament-bone and other tissues. (e.g. dentin-
enamel). Conventional Biomaterials are monophasic or composite materials
are inefficient facilitating tissue formation. So, gradient materials are required
for interface tissue engineering.
■ Gradient biomaterials will serve as extracellular matrix and provide
temporary support to grow and organize into functional tissues. This will
regulate most cell behavior such as alignment, motility, differentiation,
mitosis and other critical bio logical process include immune response,
embryogenesis and interface tissue formation.

3. Therefore, synthetic scaffolds made of
gradient biomaterials have numerous
advantages over their conventional
monophasic material but preparation
and characterization are generally
more difficult.
Recent advances in micro and Nano
engineering approaches with gradient
in material properties that favor the
culture end growth of stem cells.
Particularly with regard to cell
differentiation which is a necessary
step toward the development of
tissues suitable for interfacial tissue
engineering.

4. ■ Gradient biomaterials are classified into
various types.
■ (1) Material composition (e.g. different
polymer concentration or composition).
■ (2) Material structures (e.g. gradient
thickness or porosity), the physical and
mechanical properties of the material (e.g.
gradients of wettability or stiffness) and
the interaction of the material with cells
(e.g. cross-gradients of adhesive and
non-adhesive polymers).
■ Apart from all these it can be described
with materials coating. (e.g. gradient of
adhesive peptides) or by incorporating a
soluble or immobilized molecular factor or
drug into the biomaterials.
CLASSIFICATION

5. PHYSICAL GRADIENT
■ Materials with graded variation in their physical properties including porosity,
stiffness and topography. In structure of ligament-bone, cartilage-bone or
tendon-bone interface mechanical properties of one tissue into the
mechanical properties of the other tissue via a gradual change in structural
organization and nature of tissue.
■ Porosity and pore size are very important features of a tissue scaffold that
greatly affect cell behaviors, particularly cell adhesion, migration, proliferation
and phenotype expression. For example endothelial cells showed
proliferation with 5cem pore size, hepatocytes preferred 20µm, fibroblasts 90-
360µm and osteoblast 100-350µm pore size.

6. ■ Cells form a mixture of chondrocytes, osteoblasts and fibroblasts cultured on a
bore size gradient colonized in different areas depending on the size of pores.
Biochemical gradient in the body is tendon-bone interface where the stiffness
of the bone gradually converts to the elasticity of the ligaments. Stiffness exists
because cells can precisely sense physical stress and adjust the rigidity of their
cytoskeleton as their traction force at their anchoring site.
■ Surface gradients in term of roughness, hydrophilicity and crystallinity have a
strong effect on cellular adhesion, spreading, proliferation and ECM deposition.
Washburn et.al. introduced a roughness gradient from 0.5 to 13mm on a poly (l-
lactic acid) film and studies the effect of surface roughness with pre-
osteoblastic MC3T3-EI cells. This study demonstrated that cells respond to
roughness and that the cell density decrease with increasing roughness.

7. CHEMICAL GRADIENT
■ Biomaterials with chemical gradients are referred as materials with gradients of
chemical functionalities or properties. Many of studies with wettability gradients
have focused on cell adhesion and spreading. In some cases, a spacer has been
used between the substrate and active molecules. Liu et.al formed a gradient of
C11OH SAM on a gold layer substrate using electrochemical desorption, backfilled
the spaces with C15COOH and then activated the carboxyl group to fix adhesive
protein molecule . Such as fibronectin(FN) on growth factors such as VEGF.
■ The cells moved faster toward the protein gradient when graded surface were
loaded with bovine aortic endothelial cells (BAECs) compared with the uniform
control surface and the effects of multiple gradients were cumulative.

8. BIOLOGICAL GRADIENT
■ Proteins are biological molecules are involved in Biological gradients. The
generation of gradient with adhesive peptide and natural ECM protein to study the
cellular functions with improve biomaterial properties.
■ The arginine-glycine-aspartic acid (RGD) motif is a sequence found in native
ECM proteins, such as fibronectin, fibrinogen and laminin that acts as a cell
adhesion ligand with integrins.
■ RGD is often used to enable cell attachment to fibroblasts cultured on a PEG
hydrogel with an immobilized RGD gradient aligned and moved along the gradient.

9. MICRO AND NANO ENGINEERING TECHNIQUES FOR
FABRICATING GRADIENT BIOMATERIALS
■ Scaffold should mimic the
structure and function of native
ECM, in which cells and tissue
are organized into 3D
architectures and are triggered
by various signaling to support
cell adhesion, proliferation end
differentiation.

10. SALT LEACHING
■ Salt is used to create pores or channels in the 30 polymeric scaffolds. In this
procedure casting of the mixture of the polymer, salt and organic solvent into a
mold. After solvent evaporation, salt particles are leached away with water to
generate a scaffold. Wu et.al developed a poly(L-Lactic acid)(PLLA) scaffold by
Nacl particle leaching. The scaffold was placed vertically in a beaker and then
aminolyzed along a gradient by wetting it at a controlled speed from bottom to top
with 1,6-hexanediamine –propane solution. Gelatin was then immobilized by the
amino groups via a glutaraldehyde coupling agent to form a gelation gradient.

11. GAS FOAMING
■ In gas foaming, a polymer phase is
saturated with gas such as CO2 at
high pressure(800 psi).When the
pressure inside chamber is quickly
released, gas bubbles are
generated and grow in the
polymer, a process called foaming.
After complete foaming process,
the polymeric scaffold turns into a
3D scaffold. The amount of co2
determine the porosity.

12. PHASE SEPARATION
■ In phase separation , a
homogenous polymer solution into
a polymer-lean phase and polymer
rich phase due to the addition of
immiscible solvent or to a decrease
temperature below the solvent
melting point. Subsequent freeze
dying of the liquid-liquid phase
results in solvent removal and
produces micro porous structures.

13. EMULSIFICATION
■ In emulsification, a polymer is
dissolved in an organic solvent
followed by water addition and the
two phases are stirred to obtain
an emulsion. The emulsion then
poured into liquid nitrogen followed
by freeze-drying to remove
disperse water and solvent, giving
scaffold a porous structure.

14. ■ SFF can be formed by
delivering energy or
materials to a specific
points. It includes electron
beam melting, fused
deposition modeling, stereo
lithography , laminated
object modeling, selective
laser sintering and 3D
printing.
SOLID FREE FORM TECHNOLOGY

15. PHOTOLITHOGRAPHY
■ Photoresist polymer
undergoes selective
photo polymerization
caused by selective
uv irradiation through
a photo mask with
the desire pattern
geometry.

16. ■ Micro channels are used to
deliver fluids to selected areas
of substrate and substrate
expose to flow, resulting,
pattering of the material.
MICROFLUIDICS

17. MICRO CONTACT
PRINTING
■ It allows for the transfer
of patterns onto
biomaterial substrate
with high spatial
resolution suitable for
cell studies.

18. ■ Ejection of a polymeric jet
from the tip of an electrically
charged syringe, the
spinneret followed by its
collection onto a counter
electrode resulting the
formation of fiber with size
usually ranging from 10 nm to
few micrometers.
ELECTROSPINING

19. NANOIMPRINT LITHOGRAPHY
■ A thermoplastic or UV-curing
polymer layer is imprinted by a mold
and cured by heat or by UV
irradiation at the same time. After
cooling down UV the stamp is
removed which contains reverse
stamp topography.

20. ■ Inkjet printing is a noncontact
reprographic method that
translates numerical data from
computer into a pattern on a
substrate using ink drops.
INKJET PRINTING

21. GRADIENT MARKERS
Gradient is formed
because the solution from
chamber A is mixed with a
decreasing volume of
solution from chamber B.

22. CONCLUSION
■ The development of biomaterials with gradient in mechanical properties ,
composition, structure or incorporated biomolecule is essential. Micro and
nanotechnologies allow for the fabrication of such gradient biomaterials and can
be used to create new, advanced gradient biomaterial for ITE application.
Electrospun scaffold for stem cells
and tissue regeneration