Biomaterials immobilization:

Biomaterials are materials that are used in contact with biological systems. Biocompatibility
and applicability of surface modification with current uses of metallic, polymeric and ceramic
biomaterials allow alteration of properties to enhance performance in a biological
environment while retaining bulk properties of the desired device.
Surface modification involves the fundamentals of physicochemical interactions between the
biomaterial and the physiological environment at the molecular, cellular and tissue levels
(reduce bacterial adhesion, promote cell adhesion). Currently, there are various methods of
characterization and surface modification of biomaterials and useful applications of
fundamental concepts in several biomedical solutions.

Functions:
The function of surface modification is to change the physical and chemical properties of
surfaces to improve the functionality of the original material. Protein surface modification of
various types biomaterials (ceramics, polymers, metals, composites) is performed to
ultimately increase biocompatibility of the material and interact as a bioactive material for
specific applications. In various biomedical applications of developing implantable medical
devices (such as pacemakers and stents), surface properties/interactions of proteins with a
specific material must be evaluated with regards to biocompatibility as it plays a major role in
determining a biological response. For instance, surface hydrophobicity or hydrophilicity of a
material can be altered. Engineering biocompatibility between the physiological environment
and the surface material allows new medical products, materials and surgical procedures with
additional biofunctionality.
Surface modification can be done through various methods, which can be classified through
three main groups: physical (physical adsorption, Langmuir blodgett film), chemical
(oxidation by strong acids, ozone treatment, chemisorption, and flame treatment) and
radiation (glow discharge, corona discharge, photo activation (UV), laser, ion beam, plasma
immersion ion implantation, electron beam lithography, and γ-irradiation).

Biocompatibility :
In a biomedical perspective, biocompatibility is the ability of a material to perform with an
appropriate host response in a specific application. It is described to be non-toxic, no induced
adverse reactions such as chronic inflammatory response with unusual tissue formation, and
designed to function properly for a reasonable lifetime.  It is a requirement of biomaterials in
which the surface modified material will cause no harm to the host, and the material itself
will not harmed by the host. Although most synthetic biomaterials have the physical
properties that meet or even exceed those of natural tissue, they often result in an unfavorable
physiological reaction such as thrombosis formation, inflammation and infection.
Biointegration is the ultimate goal in for example orthopedic implants that bones establish a
mechanically solid interface with complete fusion between the artificial implanted material
and bone tissues under good biocompatibility conditions. Modifying the surface of a material
can improve its biocompatibility, and can be done without changing its bulk properties. The
properties of the uppermost molecular layers are critical in biomaterials since the surface
layers are in physicochemical contact with the biological environment.
Furthermore, although some of the biomaterials have good biocompatibility, it may possess
poor mechanical or physical properties such as wear resistance, anti-corrosion, or wettability
or lubricity. In these cases, surface modification is utilized to deposit a layer of coating or
mixing with substrate to form a composite layer.
Cell adhesion:
As proteins are made up of different sequences of amino acids, proteins can have various
functions as its structural shape driven by a number of molecular bonds can change. Amino
acids exhibit different characteristics such as being polar, non-polar, positively or negatively
charged which is determined by having different side chains. Thus, attachment of molecules
with different protein for example, those containing Arginine-Glycine-Aspartate (RGD)
sequences are expected to modify the surface of tissue scaffolds and result in improvement of
cell adhesion when placed into its physiological environment. Additional modifications of the
surface could be through attachment of functional groups of 2D or 3D patterns on the surface
so that cell alignment is guided and new tissue formation is improved.
Biomedical materials :
Some of the surface modification techniques listed above are particularly used for certain
functions or kinds of materials. One of the advantages of plasma immersion ion implantation
is its ability to treat most materials. Ion implantation is an effective surface treatment
technique that be used to enhance the surface properties of biomaterials. The unique
advantage of plasma modification is that the surface properties and biocompatibility can be
enhanced selectively while the favorable bulk attributes of the materials such as strength
remain unchanged. Overall, it is an effective method to modify medical implants with
complex shape. By altering the surface functionalities using plasma modification, the optimal
surface, chemical and physical properties can be obtained.
Plasma immersion implantation is a technique suitable for low melting point materials such
as polymers, and widely accepted to improve adhesion between pinhole free layers and
substrates. The ultimate goal is to enhance the properties of biomaterials such as
biocompatibility, corrosion resistance and functionality with the fabrication of different types
of biomedical thin films with various biologically important elements such as
nitrogen, calcium, and sodium implanted with them. Different thin films such as titanium
oxide,[18] titanium nitride, and diamond-like carbon have been treated previously, and results
show that the processed material exhibit better biocompatibility compared to the some current
ones used in biomedical implants. In order to evaluate the biocompatibility of the fabricated
thin films, various in vitro biological environment need to be conducted.

Biological response
The immune system will react differently if an implant is coated in extra-cellular matrix
proteins. The proteins surrounding the implant serve to "hide" the implant from the innate
immune system. However, if the implant is coated in allergenic proteins, the
patient's adaptive immune response may be initiated. To prevent such a negative immune
reaction, immunosuppressive drugs may be prescribed, or autologous tissue may produce the
protein coating.
Acute response
Immediately following insertion, an implant (and the tissue damage from surgery) will result
in acute inflammation. The classic signs of acute inflammation are redness, swelling, heat,
pain, and loss of function. Hemorrhaging from tissue damage results in clotting which
stimulates latent mast cells. The mast cells release chemokines which activate blood vessel
endothelium. The blood vessels dilate and become leaky, producing the redness and swelling
associated with acute inflammation. The activated endothelium allows extravasation of blood
plasma and white blood cells including macrophages which transmigrate to the implant and
recognize it as non-biologic. Macrophages release oxidants to combat the foreign body. If
antioxidants fail to destroy the foreign body, chronic inflammation begins.
Chronic response[edit]
Implantation of non-degradable materials will eventually result in chronic inflammation and
fibrous capsule formation. Macrophages that fail to destroy pathogens will merge to form
a foreign-body giant cell which quarantines the implant. High levels of oxidants cause
fibroblasts to secrete collagen, forming a layer of fibrous tissue around the implant.
By coating an implant with extracellular matrix proteins, macrophages will be unable to
recognize the implant as non-biologic. The implant is then capable of continued interaction
with the host, influencing the surrounding tissue toward various outcomes. For instance, the
implant may improve healing by secreting angiogenic drugs.

Fabrication techniques :

Physical modification
Physical immobilization is simply coating a material with a biomimetic material without
changing the structure of either. Various biomimetic materials with cell adhesive proteins
(such as collagen or laminin) have been used in vitro to direct new tissue formation and cell
growth. Cell adhesion and proliferation occurs much better on protein-coated surfaces.
However, since the proteins are generally isolated, it is more likely to elicit an immune
response. Generally, chemistry qualities should be taken into consideration.
Chemical modification

Covalent binding of protein with polymer graft

Alkali hydrolysis, covalent immobilization, and the wet chemical method are only three of
the many ways to chemically modify a surface. The surface is prepped with surface
activation, where several functionalities are placed on the polymer to react better with the
proteins. In alkali hydrolysis, small protons diffuse between polymer chains and cause
surface hydrolysis which cleaves ester bonds. This results in the formation of carboxyl and
hydroxyl functionalities which can attach to proteins. In covalent immobilization, small
fragments of proteins or short peptides are bonded to the surface. The peptides are highly
stable and studies have shown that this method improves biocompatibility. The wet chemical
method is one of the preferred methods of protein immobilization. Chemical species are
dissolved in an organic solution where reactions take place to reduce the hydrophobic nature
of the polymer. Surface stability is higher in chemical modification than in physical
adsorption. It also offers higher biocompatibility towards cell growth and bodily fluid flow.
Photochemical modification
 Cell adhesion for various functional groups. OH and
CONH2 improve surface wetting compared with COOH
Successful attempts at grafting biomolecules onto polymers have been made using
photochemical modification of biomaterials. These techniques employ high energy photons
(typically UV) to break chemical bonds and release free radicals. Protein adhesion can be
encouraged by favorably altering the surface charge of a biomaterial. Improved protein
adhesion leads to better integration between the host and the implant. Ma et al. compared cell
adhesion for various surface groups and found that OH and CONH 2 improved
PLLA wettability more than COOH.
Applying a mask to the surface of the biomaterial allows selective surface modification.
Areas that UV light penetrate will be modified such that cells will adhere to the region more
favorably.
Composites and graft formation;
Graft formation improves the overall hydrophilicity of the material through a ratio of how
much glycolic acid and lactic acid is added. Block polymer, or PLGA, decreases
hydrophobicity of the surface by controlling the amount of glycolic acid. However, this
doesn't increase the hydrophilic tendency of the material. In brush grafting, hydrophilic
polymers containing alcohol or hydroxyl groups are placed onto surfaces through
photopolymerization.
Plasma treatment.
Plasma techniques are especially useful because they can deposit ultra thin (a few nm),
adherent, conformal coatings Glow discharge plasma is created by filling a vacuum with
a low-pressure gas (ex. argon, ammonia, or oxygen). The gas is then excited using
microwaves or current which ionizes it. The ionized gas is then thrown onto a surface at a
high velocity where the energy produced physically and chemically changes the surface.
After the changes occur, the ionized plasma gas is able to react with the surface to make
it ready for protein adhesion. However, the surfaces may lose mechanical strength or
other inherent properties because of the high amounts of energy.
Several plasma-based technologies have been developed to contently immobilize proteins
depending on the final application of the resulting biomaterial. This technique is a
relatively fast approach to produce smart bioactive surfaces.

Applications
Bone tissue
Extra-cellular matrix (ECM) proteins greatly dictate the process of bone formation—the
attachment and proliferation of osteogenitor cells, differentiation to osteoblasts, matrix
formation, and mineralization. It is beneficial to design biomaterials for bone-contacting
devices with bone matrix proteins to promote bone growth. It is also possible to
covalently and directionally immobilize osteoinductive peptides in the surface of the
 ceramic materials such as hydroxyapatite/β-tricalcium phosphate to stimulate osteoblast
differentiation and better bone regeneration RGD peptides have been shown to increase
the attachment and migration of osteoblasts on titanium implants, polymeric materials,
and glass. Other adhesive peptides that can be recognized by molecules in the cell
membrane can also affect binding of bone-derived cells. Particularly, the heparin binding
domain in fibronectin is actively involved in specific interaction with osteogenic cells.
Modification with heparin binding domains have the potential to enhance the binding of
osteoblasts without affecting the attachment of endothelial cells and fibroblasts.
Additionally, growth factors such as those in the bone morphogenic protein family are
important polypeptides to induce bone formation. These growth factors can be covalently
bound to materials to enhance the osteointegration of implants.
Neural tissue
Peripheral nervous system damage is typically treated by an autograft of nerve tissue to
bridge a severed gap. This treatment requires successful regeneration of neural tissue;
axons must grow from the proximal stump without interference in order to make a
connection with the distal stump. Neural guidance channels (NGC), have been designed
as a conduit for growth of new axons and the differentiation and morphogenesis of these
tissues is affected by interaction between neural cells and the surrounding ECM. Studies
of laminin have shown the protein to be an important ECM protein in the attachment of
neural cells. The penta-peptide YIGSR and IKVAV, which are important sequences in
laminin, have been shown to increase attachment of neural cells with the ability to control
the spatial organization of the cells.
Cardiovascular tissue
It is important that cardiovascular devices such as stents or artificial vascular grafts be
designed to mimic properties of the specific tissue region the device is serving to replace.
In order to reduce thrombogenicity, surfaces can be coated with fibronectin and RGD
containing peptides, which encourages attachment of endothelial cells. The peptides
YIGSR and REDV have also been shown to enhance attachment and spreading of
endothelial cells and ultimately reduce the thrombogenicity of the implant

Biomaterials in cell growth and wound healing

Skin plays a key role in protecting our internal environment from the external environment,
maintaining homeostasis, and regulating temperature. On the outer side is the epidermis that
consists predominantly of keratinocytes, which form a tight seal for protection (Fig. 1), along
with melanocytes, Langerhan and Merkel cells . Below this is the dermis, which is attached to
the epidermis by the basement membrane, a thin layer of extracellular matrix (ECM)
consisting mostly of laminins, integrins, perlecan, nidogen, and collagen IV. The composition
of the dermis is complex and differs quite dramatically from the epidermis . It consists of
ECM, which acts as a scaffold for fibroblasts and other mesenchymal cells, blood vessels,
hair follicles, and sweat glands . It also houses molecules, such as growth factors and
enzymes, that regulate the local environment . The dermis has several sublayers, with the
papillary layer closest to the basement membrane consisting of poorly ordered thin collagen
fibers housing a high density of fibroblasts. Sandwiched between the lower dermal white
adipose tissue and the papillary layer is the reticular dermis in which collagen fibers are
thicker, more ordered, and sparsely populated with cells . This complex nature of the skin
makes it particularly difficult to replicate in the laboratory.

Fig. 1The three main layers of the skin: epidermis, dermis, and hypodermis

For many wounds, the healing process follows an ordered series of events including
homeostasis, inflammation, proliferation/matrix deposition, and remodeling (reviewed in
detail For repair to occur, fibroblasts and other cells must fill the void created by the injury,
with new blood vessels and ECM to form the granulation tissue, over which keratinocytes
migrate to reseal the skin . However, in cases such as burns where the damage to the
epidermis and dermis can be extensive, the repair process is more complex. Here, cells and
matrix to support the restoration of the skin are often reduced, or lacking, depending on the
depth and severity of the injury. This leads not only to a slow healing process but also the
potential for increased scar formation.

There is a vast number of treatments on the market for the management of wounds and burns
with the majority being wound dressings. Current wound dressings are comprised of a wide
range of material types and claims with regard to what they treat. However, questions remain
as to how well they facilitate the healing process . Wound dressings, including films and
foam dressings, are made from various materials, with some containing biologics or materials
know to have antibacterial properties or agents that can facilitate cell migration. Additionally,
there is a number of therapies currently on the market, such as skin substitutes derived from
either de-epidermized tissue that can contain skin-derived cells, or alternatively cells,
including fibroblasts and keratocytes, within a biological matrix or delivery vehicle

Review

Wound dressings

Wound dressings have been fabricated out of different types of materials and various formats,
for example fiber mats and hydrogels, and may contain additivities like silver for anti-
bacterial properties. Conventional wound dressings serve to create a sealed wound
environment to keep out infection, while also creating a moist environment to promote the
wound healing process (Fig. 2). Recent progresses in the development of advanced wound
dressings has seen the use of materials and/or the incorporation of biologics capable of either
stimulating or promoting events in wound healing, from cellular migration, to the production
of ECM components.

Fig. 2 Schematic representation of a wound dressing, designed to create a sealed wound

environment to keep out pathogens and promote the wound healing process
Fiber mats 

Conventional wound dressings were originally made from cotton gauze or non-woven blends
of similar materials. Current research into wound dressings includes electrospun mats that
create a coverage for the wound but enable the exchange of gases through the dressing. Fiber
mats prepared from polymers, including polycaprolactone, often include incorporation of a
biological material like collagen to mimic the dermis. Incorporation of known antibacterial
compounds including silver and gentamicin are an added feature of many of these dressings.

One of the drawbacks of using synthetic materials, like polycaprolactone, as a wound

dressing is that the dressing will eventually need to be removed, which may cause further
damage to the wound. Fiber mats produced from natural materials, including dermal proteins,
can be made to create wound dressings that mimic the ECM of the skin and can subsequently
be incorporated into the body. Depending on the polymer/protein used, it may also stimulate
wound healing responses. Fibronectin is one such protein found within the dermis and has
been used to make scaffolds for potential wound healing therapies, which have been shown to
not only accelerate wound healing but improve structural remodeling of the dermis and
epidermis following healing . The use of materials for the fabrication of scaffolds not only
serves as material that biologically mimics the tissue that it is replacing, but it may also
mimic the structure (Fig. 3).
Fig. 3 Scanning electron micrographs (SEMs) of the micro- and macro-structure of a native
dermal extracellular matrix (ECM) and b fibronectin scaffolds for wound healing
applications. Figure adapted with permission from the original article of Chantre et al. .
(Copyright 2018 by Elsevier Ltd)
Hydrogels 

Hydrogels (Fig. 4a) are good candidates for wound dressings as they are able to form a
barrier from pathogens, as well as create a hydrated environment to help promote the body’s
own wound healing response . Poly(vinyl alcohol) (PVA) is a polymer that is commonly used
in the fabrication of hydrogels and is frequently used in wound healing applications. PVA is
often used in medical applications as it is known for its anti-protein fouling properties and is
relatively biologically inert. PVA hydrogels for wound healing often include other materials
to stimulate the wound healing response such as curcumin or zinc oxide nanoparticles for
antibacterial properties, and phlorotannins, derived from brown algae, which have been
shown to promote fibroblast migration. A polymer similar to PVA, poly(ethylene glycol)
(PEG), is also commonly used for the fabrication of hydrogels, where Polymyxin B
conjugated to PEG has been shown to be antibacterial, and when combined as a hybrid with
alginate can promote wound regeneration . Advances in hydrogel polymerization methods
also enable the use of injectable hydrogels (Fig. 4b), which can be directly delivered onto a
patients wound enabling complete and customized coverage.

Fig. 4Various types of hydrogels have been, and are continuing to be used as wound healing
therapeutics including hydrogels formed from the biopolymer a Hyaluronan hydrogel.
Figure adapted with permission from the original article of Liyang et al.. (Copyright 2017 by
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). b Injectable hydrogels show promise
for wound healing applications.
A well-known component present in skin is hyaluronan, also known as hyaluronic acid .
Hyaluronan is a polysaccharide and is commonly used in hydrogels for wound healing.
Hydrogels composed of hyaluronic acid and chitosan have been used to deliver the
angiogenic promoting growth factor vascular endothelial growth factor (VEGF) and have
been shown to be both antibacterial and angiogenic, suggesting it might have potential as a
wound healing therapeutic. Furthermore, hydrogels that have incorporated hyaluronan have
been shown to promote blood clotting and possess antibacterial properties. Other
polysaccharides, including chitosan,alginate, and cellulose, have also been used to fabricate
hydrogels and have shown promise as wound healing therapeutics.

Wound dressings with incorporated biologics 

In each of the different phases of wound healing, various growth factors and cytokines are
involved in biological processes that result in the progression of the wound to the following
healing phase. The harsh environment within a non-healing wound often results in either the
absence of cells that produce and secrete the required growth factors and cytokines, or the
degradation of those that are present. The delivery of growth factors and cytokines to wounds
using biomaterials has been investigated not only for wound healing, but other regenerative
applications. These require not only the incorporation of the growth factors and cytokines but
also their delivery to the desired site of action in a functional and active state and at an
appropriate concentration. In vivo, many growth factors are bound and protected by
heparin/heparan sulfate including members of the fibroblast growth factor (FGF) and VEGF
families, and various cytokines that are associated with inflammation. To mimic these in
vivo interactions, heparin has been incorporated into wound healing therapeutics for the
protection and delivery of growth factors, including VEGF and transforming growth factor
beta (TGFβ) Alternate methods for incorporation of growth factors include covalent
incorporation , as well as genetically modified production of proteins to include incorporation
of growth factors or recombinant expression of growth factor fusion proteins which can then
be incorporated into biomaterial scaffolds for wound healing therapeutics. Additionally, the
incorporation of exogenous growth factors or cytokines into biomaterial scaffolds has been
shown to upregulate the expression of endogenous growth factors

Skin substitutes

There are three main types of skin substitutes available: dermal, epidermal, and
dermal/epidermal. Traditionally, skin substitutes, particularly dermal ones, have been
composed of de-epidermized tissue, leaving the ECM as a scaffold, removing any
components that could cause an immune response in recipients. More recently, different types
of skin constructs have been designed to mimic the ECM of the skin using components such
as collagen, hyaluronan, and some have skin cells incorporated into them. Several
commercially available skin substitutes, described in detail below, use xenogeneic
components for example bovine collagen. While not ideal for use in products for human use,
they are commonly used due to the lower cost, availability, and abundance as compared to
human-derived components. Technologies regarding recombinant protein production,
particularly of human origin, is becoming more common with increasing presence in the
research literature. This is likely to result in a reduction in associated costs with production
and thus be translated into clinical use in the future.

Dermal substitute 

Fibroblasts are found in every tissue of the body. In skin they are typically found embedded
in the ECM, which forms the scaffold for the dermis . Their role is to help maintain the
structure and function of the dermis by continuously secreting growth factors, ECM
precursors, and enzymes that modify these precursors. While they typically reside in the
healthy dermis, they also migrate into wounds after injury . In the injured tissue, signals in
the local environment cause fibroblasts to differentiate into myofibroblasts. One such signal
is extra domain-A fibronectin which is not usually expressed under normal conditions but is
upregulated after injury. In the wound, myofibroblasts play a key role in secreting ECM
components, such as collagen and fibronectin, which form the scaffold necessary for cells to
migrate into, and over, to populate the wound area . They also secrete growth factors, such as
platelet-derived growth factor (PDGF) that modulate other cells in the wound, and enzymes,
such as the matrix metalloproteinases and their inhibitors, that play key roles in remodeling
the ECM and contribute to the final wound healing outcome. These same myofibroblasts are
also responsible for the contractility of scar tissue as it matures .

The role of myofibroblasts in the production and remodeling of the ECM, and in the
contraction that drives fibrotic disease has led to extensive research into the nature and source
of these cells. In skin, there are at least three populations of dermal fibroblasts that can
exhibit different phenotypes depending on the location and age of the skin. The papillary
(superficial) dermal fibroblasts are found in the ridge-like structure of the papillary dermis.
Below this are the reticular dermal fibroblasts and lastly there is a population that
accumulates around hair follicles It should also be noted that dermal fibroblasts are not the
only sources of myofibroblasts in the wound, for example mesenchymal stem cells found in
the dermal sheath surrounding the hair follicle can also differentiate into wound
myofibroblasts .

Given their role in secreting ECM products that build the scaffold for cells to repopulate the
wound, it is not surprising that several skin substitutes contain fibroblasts, either from the
patients themselves (autologous) or allogenic (neonatal) fibroblasts. How well these
recapitulate the different types of fibroblasts found in the skin is unknown and as further
research into the area develops, the efficacy of these skin substitutes will improve.