Bioactive Nanoparticle Materials for Bone Tissue Regeneration (BioE 505 -- NanoBioTechnology)

1. Bioactive Scaffolds for Bone Tissue
Regeneration: Emerging Nano-materials
Final Report
Prepared by:
Kathleen Broughton
NanoBioTechnology
BIOE 505
April 13, 2010

2. Outline
Bioactive scaffold material is becoming more important and is on the cutting edge of
research today because of the foreseeable need for bone tissue regeneration as an
effective way to improve the current medical practice of bone replacement. Bone
regeneration is more effective because there are no concerns of the body rejecting the
prosthesis, less concern regarding contamination in a surgery leading to infection as
well as less patient rehabilitation.
To design a good bioactive scaffold there are a number of considerations that must
be taken into account. There has been a good deal of research at the micro-scale level
on this subject and research is now gearing itself to study nano-materials, which will be
valuable in the upcoming year(s) when an optimal material(s) is discovered.
The focus of this report is on porous nano-materials used in the search of the best
material available to assist in bone tissue regeneration. The paper will first discuss the
need for nano-materials and provide background information with respect to bone
formation. The discussion will then shift to background information about polymer and
ceramic materials studied. This foundation will allow for an informative discussion of
ceramic materials, which is where the bulk of nano-material research is conducted
today. The main comparison made in this paper is focused on comparing the porosity
to the mechanical (compressive) strength of different materials. A brief analysis of an in
vivo discussion will then be provided for proof of concept of bone tissue regeneration.
The topic and materials discussed is very current and provides an excellent opportunity
to aid in the development of this research.
2

3. I. Introduction
I.I. General Overview
The medical sciences continue to advance in parallel with improvements to the care
and methods to treat an aging population because of technological innovation. One of
the ever growing needs for our aging society is that of bone replacement and
regeneration. Tissue begins to decay progressively in humans starting around age
thirty. The National Center for Health Statistics (NCHS) reported 1,039,000 bone
fractures for all sites in 2004 in the U.S alone. Additionally, around 118,700 patients had
osteoarthritis and associated disorders in 2000. The American Academy of Orthopedic
Surgeons reported that in just a 4 year period, there was an 83.72% increase in the
number of hip replacements performed from nearly 258,000 procedures in 2000 to
474,000 procedures in 2004 [1]. The orthopedic market was estimated to be worth
approximately US $37.1 billion in 2008, following a growth of 9.7 percent over the
previous year [2]. Furthermore, the development of sturdy prostheses has not advanced
beyond the medical instrumentation to maintain life. In other words, patients are now
outliving their replacement parts; studies have shown that there is a 15 – 50% failure
rate to prostheses over a 15 – 30 year life span [3]. These statistics have driven the
medical community to research bone tissue regeneration as an alternative and better
way of replacing bone tissue loss.
Medical technology continues to make great strides in tissue replacement through
allographs (donor replacement) and autographs (self-donor replacement).
Replacement of aged, diseased or damaged tissues is more common today because of
reliable and affordable biomaterials and the perfection of surgical procedures for
prostheses implantation and subsequent rehabilitation. However, bone replacement is
susceptible to prosthesis implant rejection, transmission of diseases associated with the
transplant, shortage of available donors, continued physical therapy, and a high
financial investment compared to regeneration [4]. Therefore, regenerative bone tissue
implantation is a beneficial alternative to replacing the bone tissue through prosthesis.
3

4. To regenerate bone tissue, the body relies on scaffolds. Scaffolds are a type of
template the body uses to regenerate tissue, particularly bone tissue. The body is very
good at healing and regenerating itself when the defects are small. However, the body
cannot heal larger defects without the use of an aid. There are multiple criteria for bone
regeneration scaffolds which include: bioactivity (ability to bond to bone), osteogenic
(stimulation of bone growth), biocompatible (induce minimal toxic or immune response
in vivo), resorb safely and effectively in the body, similar mechanical properties to bone
(such as load absorption), ability to shape to a wide range of defect geometries, and
meet all regulatory requirements for clinical use [5]. In terms of the osteogenic
materials, the material should be osteoinductive (capable of promoting the
differentiation of progenitor cells down an osteoblastic lineage), osteoconductive
(support bone growth and encourage the ingrowth of surrounding bone), and
osteointegrate (integrative to the surrounding bone) [6]. Since bone tissue is the most
commonly replaced organ of the body, the necessity to engineer the proper scaffold
material is of a growing need.
Over the past decade, great strides have taken place in medical science to find an
adequate material to serve substitutionally and promote regeneration. Most scaffold
materials designed are either a porous matrix or a fibrous mesh to permit tissue in-
growth and the development of vessels for nutrient delivery. Many investigations have
taken part from both synthesis and material perspectives in aid of discovering a material
that fits all parameters of natural bone. The most difficult parameters to balance are the
mechanical strength of the material under a compression load against the porosity and
the resorbability of the scaffold material.
I.II Method of Analysis
Cancellous bone, the spongy internal bone with an open porous network, naturally
has strong compressive strength and simultaneously good porosity for nutrient
absorption. Approximately 70% of natural bone is composed of hydroxyapatite particles
( (HA) and the remaining 30% is an organic matrix, mainly collagen type
I. The structure of the bone is hierarchically organized from a macro to micro to nano-
4

5. scale. The cancellous bone has been measured to have a compressive strength of
4000 kPa when 70% porous and still maintain 200 kPa of pressure when 90% porous
[7]. This wide range of porosity to compressive strength is quite amazing and difficult to
synthesize. The resorbability of the filler material is controlled through two different
means [8]. In the first approach, the geometry of the material is optimized; In other
words, the pores are balanced against the compressive strength. In the second
approach, the chemistry is modified in the material choice. The manner in which a
material is fabricated impacts its performance as a biomaterial and, as such, the focus
of this paper is a comparison of different materials (nano and micro) in terms of porosity
and mechanical strength. [author note: there is a wide array of fabrication methods
used for scaffold material; such discussion is not of primary focus of this report. A brief
discussion of the fabrication methods with the strengths and weaknesses of such
methods along with an outline of the different material’s optimized fabrication method is
available in the appendix.]
To calculate the porosity of a scaffold, a researcher could take a variety of different
formulas to reach the same conclusion. A general formula is based on weight
measurement according to:
Where is the total porosity content (% vol.), is the measured weight of the
scaffold and is the theoretical one obtained by multiplying the scaffold volume and
the material density [8].
To calculate the compressive strength of a material, a general formula should be
applicable based on the equation:
Where σ is the stress (known as compressive strength of a material when the force
applied is perpendicular to the stress plane), F is the force penetrated into the material
and A is the surface area of the material that was impacted by the force The applied unit
system in bone tissue engineering research is consistent with utilization of standard
5

6. international (SI) units. Therefore, stress is measured in Pascals or Newtons per meter
squared. [http://www.engineersedge.com/material_science/stress_definition.htm].
An additional formula, not heavily analyzed in the thesis because it is not
consistently reported like porosity and compressive strength, is based on the
degradation of the material and is based upon the formula:
Where is the initial weight of the sample, and is the dry weight of the sample
after it was in simulated body fluid for a certain period of time [10].
I.III Thesis Theme
Over the past few decades research has been underway to find materials that will
serve the body towards self-regeneration of bone. To create regeneration in the bone,
research has focused on biomaterials – a substance that has been engineered to take a
form, which independently or with the interaction of the system, is used to direct, by
control of interactions with components of living systems, the course of any therapeutic
or diagnostic procedure, in human or veterinary medicine [8]. In other words,
biomaterials are intended to interface with biological systems to augment, repair, or
replace any tissue, organ or function in the body. The key characteristic of biomaterials
is its ability to remain in the biological environment without damaging its surroundings or
itself. Biomaterials are made from polymers, ceramics, or a composite.
This report focuses on three main conceptual understandings: (1) the need for nano-
scale scaffold materials appropriate for bone tissue regeneration, (2) the previous
research conducted with materials that utilize nano-particles, with a particular focus on
the porosity and mechanical strength of the various materials and (3) hypothesizing
what direction research is likely to proceed to find a bone tissue regenerative nano-
material. [author’s note – research related to: carbon nano-tubes and nano-fibres,
filler materials used for teeth regeneration is outside the scope of this paper.]
6

7. Figure 1: Snapshot of different nano-materials available for bone tissue regeneration (A) Scanning
electron microscopy (SEM) image of poly(L-lactic acid) (PLLA) nanofibrous scaffold with interconnected
spherical macropores created by a phase-separation technique. (B) Electrospun polycaprolactone/
hydroxyapatite/gelatin (PCL/HA/gelatin, 1:1:2) nanfibres which significantly improved osteoblast functions
for bone tissue engineering applications. (C) Densely aligned single wall carbon nanotube (SWCNT)
forest grown with novel water-assisted chemical vapor deposition in 10 min. (D) Transmission electron
microscopy (TEM) image of monodispersed magnetic Fe3O4 nanoparticles (6 nm) deposited from their
hexane dispersion and dried at room temperature. [11].
II. Need for Nano-based Materials
Research for scaffold materials that cause regeneration in bone has become more
aggressive in the last few years (months!). The need to investigate scaffold material at
the nano level is necessary because the previous attempts, at the micro level, have
been proven unfruitful to find a material that possesses the same characteristics of
natural bone. Nanotechnology has made significant advances over microtechnology in
this field of research. Nano-materials, which are materials with basic structural units,
grains, particles, fibers, or other constituent components smaller than 100nm in at least
one dimension, have become more prevalent in research the last few years. The
7

8. fundamental reason why nano-materials are important in bone tissue regeneration is
based, primarily, in a bottom-up approach to nature’s method of growth and replication.
II.I. Biology of Bone
Natural bone is a complex inorganic-organic nano-composite material, where HA
nanocrystallites and collagen fibrils are organized. The main way to produce artificial
biomaterials for bone substitution is to introduce calcium phosphate nanocrystallites,
such as HA, into a polymer matrice, such as chitosan. Composite materials are,
therefore, a leading way to produce a biocompatible material that will aid the bone in
regeneration. To understand the material selection, it is important to understand the
fundamentals of bone regeneration and how bone develops.
The fundamental thought of using materials for bone regeneration rather than bone
replacement with bioactive bonding was stated by a J. Wilson et al. in 1992 after
reporting that new bone had colonized on the surface of a monkey jaw while using
bioactive glass to replace periodontal-diseased bone [3]. This phenomenon was
labeled as osteoproduction and was soon seen by other researchers with similar
materials. A second key in developing the base of bone regeneration was the finding of
Xynos et al. that the ionic dissolution products released from the bioactive material
influenced the osteogenic precursor of the cells and the cell reproduction. This element
in the development focused on the slow degregation rate of the material and the found
Si and Ca ions that were slowly released from the material during osteostimulation. The
third key in developing the base of bone tissue regeneration is focused on the
concentrations of the ionic dissolution products that activate or up-regulate certain
families of genes in osteogenic cells. These studies were confirmed by a collaboration
of L.L. Hench, the founder of discovering the bioactivity of certain glass, and Professor
Dame Julia Polak.
The general process of osteoprogenitor cell reproduction is similar to all types of cell
reproduction. As shown below the cell completes multiple phases for bone regeneration
[3]. The first step is labeled G1 where the osteoblasts are synthesizing phenotypic
specific cellular products. A full functioning osteoblast produces osteocalcin and
8

9. tropocollagen macromolecules, which self-assemble into type I collagen, the
predominant collagnous molecule present in the bone matrix and numerous other
extracellular matrix proteins. Osteocalcin is a bone extracellular maxtrix non-
collagenous protein produced by mature osteoblasts with a synthesis that correlates
with the onset of mineralization, a critical feature of new bone formation. Following the
G1 cycle, the cell enters the S phase where DNA and RNA synthesize. Once the
sequencing of the acids is complete, the cell prepares to undergo mitosis in phase G2.
The cell goes through a self-check at this time to monitor cell mass and DNA/RNA
sequencing. If the chemical environment of the cell is not acceptable the cell will go
through apoptosis, die. If the cell passes its self-programmed test to specifications it
goes through mitosis, the M phase, and forms two descendant osteoblast cells. This
process is then repeated to form new generational cell replications. The self-regulation
of the cells, however, result in fewer replications with continued mitosis phases.
Therefore, the cumulative effect is a progressive decrease of bone density with time.
Studies have found that people begin the downward trend in bone tissue regeneration
around the age of thirty. For bone regeneration to fully occur, the osteoblast cell must
undergo differentiation and form into an osteocyte, which are not capable of cell
division. Osteocytes represent the cell population responsible for extracellular matrix
production and mineralization, the final and most critical step in bone development.
Understanding the regeneration of osteoblast and osteocyte cells is valuable to
determine what materials are used and why they are used in bone tissue regeneration.
Osteoblasts are responsible for self-reproduction and to form the extracellular matrix
(ECM) of bone and its mineralization [9]. The principal products of the osteoblast are
type I collagen, which is 90% of the protein in bone, vitamin-K dependent proteins,
osteocalcin and matrix Gla protein. The product of the osteoblast is the differentiation
cycle mentioned above. Osteoblasts that form into osteocytes are responsible for
intercellular communication within the bone and also to break down the bone matrix and
release calcium. Candaliculi provides the opportunity for nutrients and oxygen to pass
between the blood vessels and the osteocytes. Osteoclasts are polarized cells and
require mineral dissolution followed by degradation to be reabsorbed into the bone.
9

10. Fig. 2. Schematic of osteogenic progenitor cell cycle leading to (1) apoptosis; (2) mitosis and cell
proliferation; or (3) terminal differentiation and formation of a mineralized osteocyte (mature bone) [3].
Fig. 3. Schematic diagram of bone structure at cellular level. [9]
Type I collagen is a major organic component of mineralized ECM. The ECM plays
an important role in the function of growth and likely involves the convergence of
intracellular signaling pathways triggered by the ECM proteins, which is important in the
regeneration process. In addition to serving as a scaffold for mineralization, the ECM
functions as a substratum for bone cell adhesion and differentiation. To regenerate
10

11. bone, the ECM plays a viable role and any material used in regeneration process
should assist the cells in the matrix. Since bone ECM is a nano-composite, both
organic and inorganic nano-materials should be considered in bone tissue engineering.
IV.I. Natural Bone Materials – nano-Hydroxyapatite and Collagen
Natural tissues and organs are composed of nano-structured ECM; nature itself is
based upon formation from the bottom-up. It is, therefore, only natural that to replicate
any cellular matrices nano-materials must be incorporated. Bone consists of a protein
based soft hydrogel template (ie. collagen, non-collagenous proteins (laminin,
fibronectin, vitronectin) and water) as well as hard inorganic components
(hydroxyapatite). Cancellous bone is 70 – 90% porous and approximately 70% HA ,
30% Collagen type I. The HA matrix is typically 20-80 nm long and 2-5 nm thick. [6].
HA is a major mineral component of calcified tissues and is considered to be a
building block where plate-like HA nano-crystals are incorporated into collagen nano-
fibres. HA-enhanced surface properties have been used to promote cell response and
proliferation to induce mineralization in bone tissue engineering, which is one of the
critical steps in bone formation [2]. HA acts as a chelating agent for mineralization of
osteoblasts in bone tissue regeneration. Collagen, the other main component of the
scaffold, supports the cells for adhesion and proliferation. Collagen and HA significantly
inhibit the growth of bacterial pathogens, the frequent cause of prosthesis-related
infection, compared with poly(lactide-co-glycolide) devices. HA, in natural bone, is a
nano-material and is in its just-beginnings of research in the form of nano-
hydroxyapatite (nHAP).
II.II. Influence of nano-materials
Beyond having a similar dimension to bone tissue, nano-materials also possess
similar surface properties such as surface topography, surface chemistry, surface
wettability and surface energy due to their significantly increased surface area and
roughness compared to conventional or micron structured materials [1]. Studies have
shown that nano-structured materials with cell favorable surface properties may
11

12. promote greater amounts of specific protein interactions to more efficiently stimulate
new bone growth compared to conventional materials. Nano-material based scaffolds
can be grown or self-assembled through processes such as electrospinning, phase
separation, self-assembly processes, sintering, and solvent casting / leaching. To
fabricate nano-material scaffolds, it is important to understand the bone’s composition.
Fig. 4. Biomimetic advantages of Nano-materials for scaffold material (A)The nano-structured hierarchal
self-assembly of bone. (B) Nanophase titanium (top, the atomic force microscopy image) and
nanocrystalline HA/HRN hydrogel scaffold (bottom, the SEM image). (C) Schematic illustration of the
mechanism by which nano-materials may be superior to conventional materials for bone regeneration.
The bioactive surfaces of nano-materials mimic those of natural bones to promote greater amounts of
protein adsorption and efficiently stimulate more new bone formation than conventional materials. [1]
Nano-materials serve as an excellent candidate for synthetic scaffold material,
particularly in composite based materials, because it replicates similar characteristics as
12

13. natural bone. Studies of polymer, ceramic, and composite materials will now be
reviewed to demonstrate why nano-materials are necessary to successfully regenerate
bone tissue.
III. Polymer Based Materials
III.I. Natural-based Polymers
There are two types of natural biodegradable polymers; polysaccharides (starch,
alginate, chitin/chitosan, hyaluronic acid derivatives) and proteins (soy, collagen, fibrin
gels and silk) [11]. Biofibers such as ligneocellulosic natural fibers are also used as
reinforcements to these polymers.
Chitosan, for example, is a good polymer for scaffold regeneration because it
degrades at a rate similar to that of new tissue formation and does not release toxins
during degradation leading to complications, such as inflammation. Chitosan has been
found to work well with natural bone and has excellent osteoconductivity and
biocompatibility. Thus, chitosan has been researched in conjunction with
hydroxyapatite (HA) and has been found to have good mechanical properties as a
composite. Further discussion of chitosan in composites is discussed below.
III.II. Synthetic-based Polymers
Synthetic polymers can be produced under controlled conditions and, therefore, are
quite predictable and reproducible in terms of mechanical and physical properties, such
as tensile strength, elastic modulus and degradation.
In terms of polymers, the most often utilized biodegradable synthetic polymers are
saturated poly-α-hydroxy esters, which include poly(lactic acid) (PLA) and poly(glycolic
acid) (PGA), and poly(lactic-co-glycolide) (PLGA) [12]. The chemical properties of
these polymers allow hydrolytic degradation through de-esterification. Upon
degradation, the monomeric components are removed naturally by the body through its
pathways. PLA and PGA can be processed easily and the degradation rates, physical
and mechanical properties are adjustable over a wide range by using various molecular
13

14. weights and copolymers. However, these polymer-based scaffolds can also fail
prematurely because of a bulk erosion process or cause a strong inflammatory flair
through an abrupt release of acidic degradation by-products, which can lead to toxicity.
Table 1. Popular Bioactive Scaffold Polymer Materials – reported in 2006 [12].
The degradation of polymers has a very large range based on the molecular
composition of the material. PLGA, for example, has a wide range of degradation rates
governed by both hydrophobic/hdydrophilic balance and crystallinity all which is based
upon the composition of the molecular chains of the material. The degradation of the
polymers mentioned thus far occurs in the bulk of the material; some polymers degrade
on the surface.
Surface bioeroding polymers undergo a heterogeneous hydrolysis process and thus
erode primarily on the surface. These polymers include poly(anhydrides, poly(prtho-
esters) and polyphosphazene. These polymers are utilized more frequently for drug-
delivery rather than bone tissue regeneration but can be used as a bulk degradable
material by using a high surface to bulk ratio in the scaffold. The use of polymers,
despite some of the difficulties, works well in a composite to serve as a scaffold material
and is discussed further below.
14

15. III.III Risk associated with Polymers
One of risk with using a synthetic polymer is its potential toxicity. While ceramic
based biodegradable materials are found within bone, polymer material is not. This
concern, of toxicity, is present not only in polymer material, generally, but also with
nano-materials. Nanotechnology research is considered too recent for any true and
accurate studies to be completed to determine the different rates of toxicology based on
nanoparticles [13]. It was suggested that nanoparticles, because of their small sizes,
could act in a manner which would modify protein structures, either by altering their
function or rendering them antigenic, thus raising their potential for autoimmune effects.
Concerns of nano-materials have been raised by both the National Science Foundation
and the Environmental Protection Agency. Currently, there is no specific regulatory
requirement to test nanoparticles. It does, however, remain heavily on the radar of
regulators. Until an understanding of the actual toxicity of nano-materials is determined,
however, research will continue to stride in this direction because the benefits to
medical science and society are greater than regulating the unknown.
IV. Ceramic Based Materials
Ceramics are well known and used for scaffold fabrication in bone tissue
engineering and are well studied because of their similar characteristics to natural bone.
As previously mentioned, cancellous bone is approximately 70% HA nanoparticles. It is
easily fitting, therefore, that a replacement material to spark regeneration is composed
of a ceramic based material. Bioceramic materials are organized into three classes:
relatively bioinert ceramics, bioactive (or surface reactive) and bioresorbable [14]. The
reactivity of bioactive ceramics in simulated body fluid (SBF) demonstrates the ability of
the material to react well to ions. A characteristic of bioactive glass and similar ceramic
materials, for example, is a kinetic surface modification that occurs to the material’s
surface upon implantation. The surface forms a biologically active hydroxyl carbonate
apatite (HCA) layer, which provides the bonding interface with tissues. The HCA phase
that forms on the bioactive implant is chemically and structurally equivalent to the
mineral phase in bone and provides interfacial bonding. The in vivo formation of an
15

16. apatite layer on the surface of a bioactive ceramic can be reproduced in a protein-free
and SBF, which has an ion concentration to nearly that of human blood plasma. [15].
The two main types of materials researched thus far, and reported below, are HA and
bioactive glass. New materials under study, such as octacalcium phosphate are also
briefly reviewed.
Table 2. Popular Bioactive Scaffold Ceramic Materials –reported in 2006 [12].
IV.I. HA / nHAP
Although bones are comprised of both collagen and carbonate-substituted HA, more
focus is on the later material because of its crystallographic properties, biocompatibility,
bioactivity and osteoconductivity [2]. Studies have been conducted in pairing nHAP to
both natural polymers and synthetic polymers. The use of nHAP is predominately done
in the form of particles or fibres and is increasing in use for a composite material
because of the improved physical, biological and mechanical properties of the scaffold
material when incorporating the nHAP. Studies conducted with the incorporation of
nHAP are discussed in the ceramics section below.
16

17. Fig. 5. SEM images of nHAP particles in form of(A) needle, (B) spherical and (C) rod shaped [16].
IV.II. Bioactive Glass
The discovery that certain glass composition has excellent biocompatibility as well
as osteoconductivity was made by Larry Hench et al. in 1969. Bioactive glass develops
a calcium-deficient, carbonated phosphate surface layer that allows it to chemically
bond to bone. This bioactivity is associated with the formation of a carbonated HA layer
on the glass surface when implanted or in contact with biological fluids [3]. The
capability of a material to form a biological interface with the surrounding tissue is
critical to bone regeneration. The fact that bioactive glass also promotes regeneration
and is biodegradable makes it a prime candidate as a bone regeneration material.
One of the predominant materials is 45S5 Bioglass®, which is composed on
45% , 24.5% , 24.5% and 6% in weight percent. It has been found
that bioactive glass surfaces can release critical concentrations of soluble Si, Ca, P and
17

18. Na ions, depending on the processing route and particle size. Furthermore, the rate of
bioresoprtion of bioactive glasses can be controlled through modifying the chemical
properties of the material. [12]. A primary disadvantage of bioactive glass, however, is
that when the material crystallizes the bioactivity significantly decreases to the extent of
becoming an inert material. Crystallization of bioactive glass occurs by viscous flow
sintering. Sintering is necessary to create dense scaffold struts that hold mechanical
stability to that of natural bone. Another drawback of bioactive glass is its low fracture
toughness and mechanical strength which provide for poor load-bearing situations.
Nevertheless, bioactive glass is a researched material because it has many favorable
attributes.
In an effort by C. Vitale-Brovarone et al., characterization research was conducted in
further developing the 45S5 bioactive glass [17]. Their team determined that pore
structure should be within a few hundred microns in range and hold porosity between 50
– 60 % to successfully replicate a scaffold material. The team utilized a sponge
impregnation technique to prepare the scaffold. In the manner of preparation, polymeric
sponges possessing an open, trabecular structure are used as a template for a ceramic
replica through impregnating the sponge with a slurry mix of ceramic powders and then
process the materials with heat. Results show the pore strut thickness from 5-20
microns and the trabecular structure closely resembles that of natural bone. The
compressive strength of about 1 ± .4 MPa was found in the samples. In the SBF, the
scaffold material surface was covered by a silica-rich layer of HA crystals. Calcium
deposits were also discovered on the scaffolds. These results add to a foundation for
further research in bioactive glass.
Another team of researchers also utilized 45S5 Bioglass® as a foundation for
researching scaffold replication and created shell scaffolds. [18]. Here, the focus is on
forming a material with high porosity and adequate mechanical properties. Although
most bioactive glass scaffolds are created by the foam replication method, this team
focused on adding organic fillers to the ceramic powder and heating, using a burning-
out method (bake out), which will improve the mechanical properties of the scaffolds but
does not change the porosity that is currently similar to that of cancellous bone. In this
18

19. method, the slurry used was prepared by dispersing the glass powders into distilled
water together with a polyvinylic binder. The optimized weight ratio of the components
was: 59% water, 29% 45S5 Bioglass®, and 12% polyvinylic binder. The aim of the
compositions was to increase the final porosity of the samples, in particular at the
surface. At the end of the process of burning-out the samples, the sponges resulted to
be coated by a thin shell made of bioactive glass and binder. The process of outer shell
coating with slurry and drying was repeated to obtain a thicker outer shell.
The scaffolds produced have an original structure with an internal structure
characterized by high porosity and thin walls with an external resistant surface similar to
a shell with strong mechanical properties. The shell scaffold can be easily handled
without damage and the porosity is sufficient to ensure excellent permeability to cells
and fluids. For the samples under review, the average total porosity calculated is about
80% vol. This value satisfies the requirements to allow bone tissue in-growth. This
team plans to follow-up with further investigation of the mechanical strength of the
material as well as its reaction to immersion in simulated body fluid.
With time, researchers have scaled down the size of the bioglass particles. One of
the studies leading to the use of nano-materials with bioactive glass was performed by
Q. Fu et al. [15]. In their study, a polymer foam replication technique was used to
prepare porous scaffolds of 13–93 bioactive glass. The 13-93 glass is approved for in
vivo use in Europe and is still in development stages in the U.S.
The elastic modulus determined from the initial linear region of the stress–stain
curve was 3.0 ± 0.5 GPa. Taking the compressive strength as the highest stress on the
stress–strain curve, the average compressive strength was 11 ± 1 MPa for eight
samples tested (porosity = 85 ± 2%). SEM images of the surface of a 13–93 glass
scaffold were taken after immersion in an SBF for 7 days. Compared to the smooth
glass surface of the as-fabricated construct, the treatment in the SBF produced a fine
particulate surface layer. High-resolution SEM images showed that the surface
consisted of a porous network of nanometer-sized, needle-like crystals. The results
19

20. suggest that the fabricated 13–93 glass scaffolds could be applied as biological
scaffolds for repair and regeneration.
Fig. 6. SEM images of the surface of a 13-93 glass scaffold after immersion for 7 days in SBF [15].
IV.II. Alternatives to HA or Bioactive Glass
Similar to HA and nHAP, calcium phosphates, as either particles or fibres, are
currently under study as a filler or coating to form the ECM. Calcium phosphates, HA
and tricalcium phosphate, has also been researched as a possible ceramic based
scaffold material. Calcium phosphates have excellent biocompatibility due to their close
chemical and crystal resemblance to bone mineral [14]. Calcium phosphates possess
osteoconductive properties and can bind bone directly under certain conditions. The
downside of HA and calcium phosphates is their slow biodegradation rate and low
mechanical strength under load-bearing stress. The dissolution of synthetic HA
depends on the type and concentration of the buffered / unbuffered solutions, pH of the
solution, degree of the saturation of the solution, and the composition and crystallinity of
the HA phase. Crystalline HA exhibits the slowest degradation rate in comparison to
other calcium phosphates. HA, compared to bone, has a better compressive strength
20

21. but weaker fracture toughness. Therefore, HA and calcium phosphates cannot be used
alone for load-bearing scaffolds but rather work well in a composite material.
A modified version of calcium phosphate recently under review is octacalcium
phosphate (OCP). It structure is similar to HA and shows better osteoblastic activity in
vivo when compared to calcium phosphate. OCP may stimulate osteoconductivity
because OCP is physicochemically converted to HA if it is implanted on bone defects
[19]. OCP also is shown to biodegrade through direct resorption by osteoclast-like cells,
which enhances the replacement of newly formed bone through progressive
implantation periods. One of the set-backs of calcium orthophosphate is its high
brittleness, which impacts the ability for the material to handle load-bearing situations.
Therefore, this material will likely only be utilized in composite bone tissue materials.
Another silicate biomaterial under research, most recently, is porous diopside
( ). The focus of the current research of C. Wu et al. has been with diopside
because of a need to find a material with adequate mechanical strength in load-bearing
situations. The previous research, as reported by Wu indicates that the problem with
and bioglass is their quick degradation rate and mechanical instability.
Therefore, their study focused on scaffold material. [4]. The scaffolds were
prepared using the polymer sponge template method where the foam template, with a
25 ppi density, was cut to the desired shape and sized to replicate a porous scaffold.
The sponge was then immersed into the slurry and compressed to merger the slurry
into the foam. The sponge was then dried at 60 degrees Celsius for one day and
sintered at 1300 degrees Celsius for three hours. The phase composition, pore
morphology and microstructure of the sintered scaffolds were characterized by X-ray
diffraction and scanning electron microscopy.
The results of this study show that show a uniform inner network structure and the
interconnectivity of the scaffolds was approximately 97% complete. Results also show
that with the increase of porosity from 75% to 90%, the compressive strength and
compressive modulus of diopside scaffolds decreased from 1360±370kPa and 68±20
MPa to 200±20KPa and 10±3.3MPa, respectively. The diopside scaffolds show stable
mechanical properties while soaking in SBF up to 14 days, but the weakened
21

22. mechanical strength was evident after 14 days of soaking. Overall, the research here
shows that diopside scaffolds possess enhanced mechanical strength and mechanical
stability and decreased degradation rate compared to bioglass and scaffolds,
which leads to their conclusion that diopside scaffolds could be a promising candidate
for bone tissue regeneration.
Although ceramics are more heavily studied as an appropriate bioactive scaffold
material for bone tissue regeneration, composite materials with ceramic are more
promising to balance all of the requisite characteristics of scaffolds. Furthermore, nano-
materials are more prevalently under investigation currently in composite materials
because of the benefits previously found in the composite based materials.
V. Composite Based Materials
There are many benefits to utilize a composite material in balancing the desirable
qualities of the chosen materials. By utilizing a composite for a scaffold material the
mechanical strength and bioactivity of a ceramic can be paired with the porosity and
degradation behavior of a polymer. Inclusion of bioactive glasses has been shown to
modify surface and bulk properties of composite scaffolds by increasing the
hydrophilicity and water absorption of the hydrophobilc polymer. Ideally, the
degradation and resorption of composite scaffolds are designed to allow cells to
proliferate and secrete their own extracellular matrix while the scaffolds gradually
disintegrate. There are a multitude of fabrication methods available to create composite
scaffolds, which are outlined in the table 1, but which discussion is beyond the scope of
this paper. The research of composite material for bone tissue regeneration, especially
with use of nano-materials, is currently in their incarnation stages.
Composite materials can offer the best qualities of each material when properly
balanced and fabricated. There are two approaches used to make a bioceramic-
polymer composite scaffold: incorporating bioceramic particles in the scaffold through a
variety of techniques and coating a polymer scaffold with a thin layer of apatite through
biomimetic processes. Factors such as polymer solution concentration, porogen type
22

23. and size, freeze-drying parameters etc play important roles in forming the desired
composite scaffold porous structure. A variety of materials have been researched in
search of an optimal composite.
Table 3. Popular Bioactive Scaffold Composite Materials – reported in 2006 [12].
V.I. Composites with HA/nHAP
The use of nHAP particles is becoming more widely used in bone tissue
regeneration because of the materials osteoconductive characteristic since the main
composition of natural bone tissue is HA particles. Since the cancellous bone material
is approximately 70% nHAP, it is intuitive to make a scaffold from this material. As
shown through the following examples, the use of a nano-scale HA particle shows
better results in terms of osteoconductivity, compressive strength and bioactivity in
comparison to micro-scale HA particles used in a composite material.
23

24. An early study, published in 2005, created a composite composed of nano-
hydroxyapatite (nHAP) and poly(lactic acid) (PLA) through the solvent-casting / salt-
leaching technique, NaCl serving as the leaching agent [16]. The average particle size
was 25 nm by 150 nm. In this early study, the researchers recognized that the nHAP
serves as a better filler material than micro-HAP because it has more homogenous
distribution in the scaffold, which directly affects the mechanical properties of the
materials. The finding of the study indicated that the nHAP particles formed varied in
size between 25 and 50 nm length by 150 – 300 nm length and were phatelet-shaped
with a uniform morphology.
Fig. 7. (a) TEM images of the nHAP particles; (b) selected area electron diffraction pattern of the HA
particles shown in (a) [16].
Further results showed that the scaffolds swelled from water absorption because
PLA is highly hydrophobic. The scaffolds, when immersed in water for 24 hours had an
increase of 540% for pure PLA and 274% when 50wt% of the scaffold was nHAP. The
porosity of the scaffold was approximately 86.2% when the wt% of nHAP was 50%;
when there was no nHAP in the scaffold, the material was ~91.5% porous. The
mechanical strength of the scaffold increased from .29 to .44 MPa from 0 – 50 wt%
nHAP in the composite. The results from this study lead to further studies involving
nHAP because the overall conclusion was that nHAP provides a solid structure for the
24

25. scaffold material and demonstrates osteoconductivity, biodegradability, and mechanical
strength that is comparable to cancellous bone.
A more recent study of nHAP with PLA was conducted by Cai et al. when the study
incorporated nHAP with chitosan in the presence of PLA [20]. The choice of chitosan
with nHAP was natural because of the chemical composition of bone. These materials
together show strong biocompatibility and also has strong regenerative efficacy and
osteoconductivity. PLA was introduced because it has a high mechanical strength and
is widely used for implants. In this study, nHAP rod-shaped particles that were
approximately 50 nm by 300 nm were homogeneously distributed into a chitosan/PLA
matrix and studied in terms of morphology and mechanical properties.
SEM results indicate that the nHAP particles have a tendency to disperse in the
chitosan, which indicates that the PLA was directing the interaction of the nHAP.
Observations also indicate that the PLA nanoparticles, which varied in size between 30
to 50 nm, were heterogeneous and rationalize why the nHAP was induced to good
osteoconductivity. This heterogeneous nucleation was also caused by the mineral
crystallinization of the calcium and phosphate ions in the materials and the bone
regenerating. The compressive strength of the chitosan/nHAP with different HA
content, ranging from 50-80%, was measured with test samples including and not
including PLA. The porosity was not measured in this study. Results indicate that a 70
wt.% nHAP content held the best compressive strength for the two sample types (with
or without PLA) and measured ~270 and 250 MPa for each sample type, respectively.
The overall consensus of the study found that nano-sized HA particles in polymers
increase the bioactivity and osteoconductivity of the composite scaffold compared to
micro-sized particles or no particles. The study conclusion also found the close
combination between the nano-sized inorganic particles and the organic matrices
enhancing the mechanical properties with still providing the proper shape and size for
tissue growth. The third conclusion made in this study was that nHAP can accelerate
the formation of bone tissue apatite in comparison to micro or no HA particle.
25

26. Another study, carried out by E. Nejati et al., combined nHAP to PLLA because of
the superior mechanical strength that is found in the composite [21]. Since natural bone
is comprised of HAP nanocrystallites, it is natural to use this material in a synthetic
composite for bone regeneration. The choice of PLLA was chosen because of its low
toxicity, good mechanical characteristics, and its predictable degradation rates. The
analysis of this study focused on the comparison of pure PLLA to composites of micro-
HAP / PLLA and nHAP / PLLA. The SEM micrographs show that needle-like nHAP
particles were distributed within the porewalls of the nano-composite scaffold and no
aggregation appeared in the pores.
The pore size of pure PLLA and nano-composite scaffolds were in the range of 167
to 95 μm, respectively. The mechanical properties indicate the average of both
compressive modulus and strength of the microcomposite (13.68 and 4.61 MPa) and
the nano-composite (14.07 and 8.46 MPa) scaffolds are statistically significant higher
than those of pure PLLA scaffolds (1.79 and 2.4 MPa). Furthermore, the compressive
strength of nanocomomposite HAP is greater than that of micro HAP when evaluated
with 50% weight percent HAP. These findings validate that nano-composites make a
better option than microcomposites for bone regeneration.
Graph. 1. Porosity content of pure PLLA, nHAP/PLLA and mPAP / PLLA scaffolds [21].
Graph 2. Compressive modulus and strength of the fabricated scaffolds [21].
26

27. The use of nano-hydroxyapatite (nHAP) has also been paired with polycaprolactone
(PCL) in a study carried out by Y. Wang et al. [22]. The decision to use PCL was based
on its biodegradability, good biocompatibility and excellent mechanical strength. As
mentioned, nHAP is representative of composition and structure similar to natural bone
and has good biocompatibility, osteoconductivity and osteoinductivity. The composite
material was fabricated by a melt-molding / porogen leaching technique using poly
(ethylene glycol) and NaCl particulate. The analysis focused on degradation after six
months of in vitro sublimation in a plasma blood solution and included review of the
weight loss, nHAP content, PCL molecular weight, morphology, and mechanical
properties of the material.
Fig. 8. SEM photographs of the MSCs cultured on the top surface of the PCL scaffold and the nHA/PCL-1
scaffold. Arrows show the spherical cell after day 1 (a, c) and the cell–cell contacts with fibrous
extracellular matrix after day 7 (b, d) [22].
A porous nHAP/PCL composite scaffold was prepared and compared to pure PCL.
The porosity of the composite was controlled to approximately 70% weight percent.
The composition analysis shows a linear relationship in the weight loss of the composite
scaffold to the decrease in the nHAP loss. Degradation of nHAP/PCL was faster than
pure PCL which may be due to nHAP/PCL having higher hydrophilicity and facilitating
27

28. water infiltration. Both types of materials displayed relatively stable mechanical
properties and stable pore interconnections. This composite showed slight mechanical
weakness to pure PCL but both scaffolds could provide sufficient compressive strength
to handle load-bearing situations. Overall, the investigation shows that in vitro
degradation of the composite remains promising after a six month submersion.
A similar study involving nHAP, biphasic calcium phosphate (BCP) and PCL was
reported this past month [23]. Here, the scaffolds were composed of BCP and a nano-
composite layer of nHAP and PCL was coated on the surface of the BCP. The study
focused on the nHAP shape and size along with the scaffold mechanical properties,
biological degradation, and osteogenic potential. The in vitro bioactivity of the material
was analyzed at 1, 3, 7, 14, and 28 days with a SBF at 200 mg/ml.
To study the nHAP mechanical property and bioactivity of BCP scaffolds, three
different types of nanoparticles were used (needle, rod and spherical shape). Figure 5
of this report shows the various types of nanoparticles used in the study. Needle
shaped nHAP have an average dimension of 25 nm by 110 nm; the spherical nHAP has
a narrower particle size of 30 nm and rod shaped particles have an average width of 17
nm and length of 41 nm. Results indicate that BCP with needle nHAP had a
compressive strength of 2.1 MPa, rod-like nHAP particle samples had a compressive
strength of .9 MPa, and spherical shape nHAP particle samples have a compressive
strength of 1.4 MPa. No coating BCP had a compressive strength of .1 MPa, micron
HA/ PCL coated BCP and .55 MPa for the PCL coated BCP sample, whereas pure HA
only had a .1 MPa strength. The osteogenesis of the nHAP needles also displayed the
best induction properties.
28

29. Graph 4. Compressive strength of samples: BCP, BCP with micro HA coating, BCP with PCL coating,
BCP with PCL/nHAP needle coating, BCP with PCL/nHAP rod coating, and BCP with PCL/nHAP
spherical particle coating. [23]
The combination of nHAP and PLGA was also researched, recently, by Y. Cui et al.
[24]. In this study, nHAP wt% ranged from 5 – 40 and the material was prepared by the
melt-molding and particulate leaching methods. The studies focused on porous
scaffolds of pure PLGA, nHAP/PLGA at various nHAP wt% and HA/PLGA at various HA
wt%. Regarding mechanical strength results, the findings indicate that 20 wt % nHAP /
PLGA had the greatest compressive strength at 2.31 MPa. The compressive strength
weakened when the nHA wt % was 40. The 10 wt % nHAP/PLGA showed higher
compressive strength than HA/PLGA but the difference was slight. The porosity of the
40 wt% nHAP/PLGA was 86; pure PLGA had a porosity of 87% and HA/PLGA had a
porosity of 88%. Surprisingly, the 10 wt% and 20 wt% nHAP/PLGA had porosity at 91
and 93%, respectively. These two samples also showed better cell proliferation after
the samples underwent seven days in SBF compared to the 40 wt% nHAP/PLGA. All
nHAP/PLGA samples show better cell proliferation than the HA/PLGA sample and pure
PLGA sample. This may be due to the homogenous spread of nHAP over HA particles
or the slower degradation rate of the nHAP materials. The absorbancy of the 10 and 20
wt% nHAP/PLGA materials also relates to the improved osteoconductivity. The study is
further discussed in the In Vivo section below.
29

30. Graph 5. Osteoblast proliferation analysis [24].
HA and nHAP are a logical material of choice in bone tissue regeneration.
Osteoconductivity is shown in bone tissue when nHAP begins to form. It is likely that
research will continue to escalate with nHAP as scientists become more articulated with
the methods of fabrication and optimization of it in a composite material.
V.II. Composites with Bioactive Glass
Bioactive glass (BAG) and bioactive glass ceramics (BGC) continue to be studied
because it is known to have better performance results than hydroxyapatite (HA). BAG
has been used as bone filler material, applied in clinical treatment of periodontal
disease, and used to replace damaged middle ear bone. This filler can serve as a
reinforcing component to enhance the stiffness of polymer composites. Compared with
micron-sized bioactive ceramic particles, nano-sized particles have a large specific
surface area and can form a tighter interface with the polymer matrix in composites.
Introduction of nano-sized BGC particles into polymeric materials can not only endow
polymer scaffolds with biomineralization capability but also increase the stiffness of
polymer material without greatly decreasing the mechanical strength [25].
30

31. A few reported studies using nano-bioactive glass ceramic particles (nBGC) were
conducted by M. Peter et al. Both of these studies focused on the inclusion of nBGC
and chitosan, a biopolymer derived from partial deacetylation of chitosan. [10, 26]. It is
reported that an alternative to tissue regenerative engineering is cell based tissue
engineering. This theory is primarily based on the studies that have proven that
nanophase ceramics, compared to microphase ceramics, have better cell-material
interactions. Chitosan is known to support cell attachment and proliferation because of
its chemical properties. The aim of both studies was to form a composite with chitosan
and improve the mechanical and biological properties of the chitosan through the
incorporation of bioceramic nanoparticles.
The analysis in one of their studies focused on chitosan gelatin (CG) and nBGC with
analysis focusing on the morphology, porosity, mechanical strength and degregation of
the material [10]. The results show the pore size of the nano-composite scaffolds varied
from150 to 300 µm, which is adequate for cell migration into the interior regions of the
scaffolds. There was also a decrease in the pore size when the chitosan concentration
increased. The degradation rate was significantly decreased with the addition nBGC
which may be due to neutralization of the acidic degradation products of chitosan by the
alkali groups leaching from nBGC, thus reducing the degradation rate of the scaffold.
The degradation is also higher in the samples that contained more CG. The bioactive
studies of the report also indicate that the nano-composite scaffolds mineralize in vitro.
No commentary was made in regard to the mechanical strength of the materials in load-
bearing situations. The overall conclusion was that GC/nBGC composite is a promising
material for bone tissue engineering.
31

32. Fig. 9. SEM micrograph showing the macroporous microstructure of CG (a and b) and composite scaffold
(c and d). Pore size ranged from 150 to 300µm [10].
The second study with nBGC and chitosan by M. Peter et al. focused on a material
that did not have the natural polymer material, gelatin, in its composition. In this study,
the composite was prepared by a blending and lyophilization technique [26]. The
prepared composite particle size was reported at 100nm. The study focused on the
swelling, density, degradation and in vitro biomineralization of the material. The swelling
is important in a pore because it can aid in the supply of nutrients and oxygen to the
interior regions of the scaffold. It was found that the swelling of the scaffold can be
controlled by the amount of nBGC material in the composite. The biodegradation on
nBGC / chitosan, compared to chitosan, is much steadier and slower over time.
Furthermore, in vitro biomineralization studies show the deposition of minerals on the
surface of the composites after seven days in SBF through XRD and SEM. Overall,
Peter et al., concluded that the use of nBGC is a valuable material to use in composite
materials for bone regeneration.
In a study by Z. Hong et al., BGC nanoparticles prepared via a three-step sol-gel
method with PLLA were found to exhibit bioactive properties [25]. The analysis of the
study included the porosity of the materials, weighed at different time intervals after
being soaked in SBF, as well as SEM, XRD, and TEM analysis of the materials.
32

33. Fig. 10. SEM morphology for the porous PLLA/BGC scaffolds with different BGC contents: at low-
magnification: (A) 0 wt.%, (B) 10 wt.%, (C) 20 wt.% and (D) 30 wt.%; at high-magnification (E) 0 wt.%, (F)
10 wt.%, (G) 20 wt.% and (H) 30 wt.%. [24].
The degradability of the PLLA/BGC nano-composite scaffolds were analyzed by in
vitro analysis is plasma body solution. The degredation of the material was monitored
by water uptake, weight loss and pH variation in the medium. It was observed that after
one day of immersion, compared with PLLA porous scaffold, the water content of all the
33

34. PLLA/BGC composites increased with the introduction of BGC nanoparticles, which
have a hydrophilic character. A mass increased of 600% is observed for pure PLLA
foam, while for PLLA/BGC composites, this increased ranged from 650 – 800%. PLLA
with 10%wt BGC composite exhibits the highest water absorption throughout the whole
incubation period. The water uptake ability of the nano-composite gradually decreases
with further increase of filler loading due to the decrease in porosity at higher filer
contents. Overall, the researchers found that the inclusion of BGC nanoparticles could
increase the water uptake of PLLA scaffolds, especially at a lower BGC weight percent,
which will also improve the degradation rate of the PLLA matrix.
Fig. 11. TEM morphology of BGC nanoparticles. Bar is 200 nm [24].
More recently, nBGC and PLA were paired in a study and reviewed by A. El-Kady et
al. in regards to the development, characterization and in vitro bioactivity of the material
[27]. Bioactive glass nanoparticles were prepared by a quick alkali-mediation method
followed by a sol-gel process to create the bioactive glass/PLA material. The particles
were controlled to the range of 20-40 nm through adjustment of the pH level in the sol
ammonia solution. The characterization of the study included the porosity, degradation,
and in vitro bioactivity of the material. The results show the density of the PLA
increased with the introduction of sol-gel bioactive glass filler and the porosity of the
composite samples decreased with the increase of the glass content. The weight loss
34

35. of the material was shown to increase with both the time soaking in SBF and with the
increase of the glass content.
Results also show that a negligible amount of calcium and phosphorous was found
on the PLA after soaking the material in SBF for thirty days. On the other hand, the
nBGC/PLA materials showed there was a layer of spherical particles on the surface but
the material was still calcium-deficient. Such deficiency could be due to the poor ability
of HA to attract silica ions because of the glass particles. Overall, the results show that
the addition of sol-gel bioactive glass nanoparticles enhanced the bioactivity of the
scaffold and improved the ability to form an HA apatite layer on the surface. A follow-up
research project is determining how to improve the mineralization of the material by
adjusting the nBGC content.
V.III. Other composite materials
The use of PCL has also received attention as a composite material when paired
with poly(lactide-co-glycolide)(PLGA). A unique design was created and tested by J.
Wang and X. Yu which mimics a natural bone structure with an inner porous spiral
interior and a rigid outer tubular part. [28]. The outer part was fabricated with PLGA
sintered microparticles and the inner part consists of nanfibre-coated highly porous thin
PCL sheets. This design allowed for cells to grow completely across the thin scaffold
walls and allow for nuturient supply and waste removal. Nanfibres were also deposited
on the surface of the spiral structured scaffold to serve as extra-cellular matrix (ECM)
mimics for cell proliferation. The incorporation of nanfibres also promotes tissue
regeneration through cell attachment, proliferation and differentiation.
There were four experiment groups reported: PLGA cylinder scaffold; PLGA tubular
scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular
scaffold with PCL nanfibre containing spiral structured inner core. The results show
some variation between the different designs but, for the most part, show very similar
35

36. characteristics regarding porosity and mechanical strength which put the design and
material composite as good candidates for bone tissue regeneration.
Fig. 12. 3-D modeling of PLGA / PCL composite scaffold [28].
Graphs 6 and 7: Mechanical properties of PLGA with variable ID: (A) Young’s modulus; (B) compressive
strength mechanical property of PLGA sintered tubular scaffolds with and without insert (ID = 2 mm) [28].
36

37. Composite materials have been heavy reviewed recently for bone tissue
regeneration because of the ability to balance the strengths of the paired polymer
material, which possess strong porous behavior and biodegradability to ceramics which
typically have better mechanical strength and better biocompatibility. Nano-materials
are continually being researched and are in infancy stages with regard to bone tissue
engineering. Multiple research projects have incorporated in vitro analysis of materials
but few projects have included in vivo analysis, which is likely due to the uncertainty of
the materials in living cells and the lack of FDA approval in many of the materials.
However, with the aggressive research conducted globally on this topic, these materials
will likely be reviewed in vivo in the near future.
VI. In Vivo Study
Although a good amount of research is conducted to determine what materials will
serve as a strong bioactive scaffold material, most studies are conducted in vitro.
These studies typically involve the submersion of the material in a simulated body fluid
or plasma solution with a review of the porosity, water uptake of the material after
designated periods of time (typically 1, 7, 14, and 28 days), ion retention / mineralization
and degradation of the material to determine if the material is hydrophilic and whether or
not it would serve as a strong candidate bone tissue regeneration material.
A few in vivo studies have been conducted with micro based materials but few
studies have been published using nano-materials, mainly because this research is very
current. One of the early reported studies involved nHAP/PLGA, conducted by Y. Cui et
al., was published in Spring ’09, which was discussed above in the composite section
[24]. In this study, the researchers found that 10 and 20 wt% nHAP/PLGA was the most
osteoconductive, although weaker in compressive strength compared to 40 wt%
nHAP/PLGA. In this study, twenty-two rabbits were used to test the different materials.
The experiment consisted of a 2.0 cm segmental defect made in the bilateral radius and
filled with the scaffold material sample and studied for 24 weeks post the operation.
The results show that an untreated defect was not naturally healing. The treated bone
defects with the experimental materials experienced various results. Bone callus
37

38. emerged at 4 weeks post-surgery in the HA/PLGA, 10 and 20 wt% nHAP/PLGA and by
week 24, all samples containing HA or nHAP were bridged by new bones. The 10 and
20 wt% nHAP/PLGA specimens were nearly filled with bone ossein at 24 weeks,
whereas the 40 wt% nHAP/PLGA were observed to have more oval-shaped
mononuclear (osteoblast) cells around the bone ossein. The pure PLGA showed the
weakest results for specimens that were filled with a bioactive material. The overall
consensus of the study indicates that nHAP/PLGA serves as a promising material for
bone regeneration.
Fig. 13. Fluorescent photographs of osteoblasts adhered on the membranes of: PLGA (A–D), 20 wt.% op-
HA/PLGA (E–H) and HA/PLGA (I–L) cultured for 1 (A, E and I), 3 (B, F and J), 5 (C, G and K) and 7 (D, H
and L) days.[24].
38

39. Fig. 14. Typical radiographs of radius resection implanted with composites: untreated control (A1&2),
PLGA (B1&2), 5 wt.% op-HA/PLGA (C1&2),10 wt.% op-HA/PLGA (D1&2), 20 wt.% op-HA/PLGA (E1&2),
40 wt.% op-HA/PLGA (F1&2) and HA/PLGA (G1&2) taken at 4 (1) and 24 (2) weeks post-surgery [24].
Another successful in vivo research project that utilized nano-material for bone
tissue regeneration was carried out and reported by Y. Liu et al. [29]. In their study, their
goal was to develop a composite scaffold that had a porous structure and similar
composition to natural bone. The team compared two different types of materials. The
first material was a gelatin / nHAP created by glutaraldehyde chemical cross-linking a
gelatin aqueous solution with nHAP granules at a 5:1 ratio. A second material using the
same gelatin / nHAP base but includes a fibrin glue (FG) mixed with recombinant
human bone morphogenetic proteins (rhBMP-2) infused into the gelatin/nHAP scaffold
and lyophilized was used.
The test subjects of the study were fourty-five adolescent New Zealand white
rabbits. After the rabbits were anesthetized, a segmental defect was made on the
middle radial diaphysis of the foot. The defect was irrigated and filled with either the
39

40. first material or second material mentioned above or nothing. The animals underwent
X-ray examinations at 4, 8, and 12 weeks.
The findings of the study showed that nHAP particles were homogeneously localized
in the gelatin walls of the gelatin/nHAP scaffold and the porosity was 91.4% with
compression strength of 1.32 MPa. The pore diameter of the gelatin/nHAP/FG scaffold
was 87.9% with a compressive strength of 1.98 MPa. A significant result shown is that
the gelatin/nHAP/FG scaffold has better bioactivity over a longer period of time and
releases at a steady rate over the course of the 40 days compared to the gelatin/nHAP
material that was completely released within half this time. This slower release allowed
for a better regeneration of the bone. Based on the radiolographs taken of the animals,
gelatin/nHAP/FG scaffold with rhBMP-2 repairs the defect better than the scaffold
material without rhBMP-2.
Graph 8. Profiles or rhBMP-2 release from gelatin/nHAP/FG scaffold and gelatin/nHAP scaffolds [29].
The study by Y. Liu is quite exciting to show the real future of nano-materials applied
in bone tissue regeneration. This study, along with others, demonstrates a wave of
opportunity in both the sciences and in the well-being of advancing the general
population. Studies should be conducted to determine how the various materials
perform when not only bone needs regeneration but also when cartilage and
surrounding ligaments and perhaps muscle need to be regenerated. Strides in this
direction will become extremely valuable to intellectual property as well as advancing
medical sciences and the general welfare of man.
40

41. Fig. 15. Radiographs of a rabbit radial bone defect repaired with different scaffolds at 4, 8 and 12 weeks.
(A–C) No graft repaired the defect. (D–F) Gelatin/nHAP/FG scaffold without rhBMP-2 repaired the defect.
(H–J) Gelatin/nHAP/FG scaffold with rhBMP-2 repaired the defect [29].
VII. Discussion
A great deal of scientific research is underway currently in the pursuit of the best
material(s) to use in bone tissue regeneration. There is a great opportunity to serve
society with solving issues related to unexpected fractures of the young, predictable
failings of old tissues, such as cancer and osteoporosis, found in the elderly and
replacing the current process of prosthetics implantation failure through this research.
Cancellous bone tissue is composed of 70% hydroxapatite nanoparticles and
approximately 30% type I collagen. Although tissue can naturally repair itself when
nano, and occasional micro, damage is done, bone tissue cannot heal itself naturally
when milli or larger damage is inflicted. Therefore, advanced research in implantation
regenerative materials are sought. The qualities desired for an optimal scaffold material
are: bioactivity (ability to bond to bone), osteogenic (stimulation of bone growth),
biocompatible (induce minimal toxic or immunie respone in vivo), resorb safely and
effectively in the body, similar mechanical properties to bone (such as load absorption),
ability to shape to a wide range of defect geometries, and meet all regulatory
41

42. requirements for clinical use. Research has been conducted on polymers, ceramics
and composites. A number of different fabrication methods have also been created and
optimized for the different materials.
The focus of this study was to: gain a general understanding of how bone tissue is
naturally formed and how nano-technology plays an important role in the development
of finding a suitable bone tissue regenerative material, understand the main materials
utilized in bone tissue regeneration, and comparing the mechanical strength to porosity
of materials that utilize nano-particles. The research conducted in this field is very
current with most of the findings by different research teams reported within the past six
to twelve months. The overall development of this type of research is proving itself to
be very beneficial with a great deal of opportunity still available to learn about the in vivo
performance of the different nano-materials utilized.
With respect to polymers, the most utilized natural materials are polysaccharides
(starch, alginate, chitin/chitosan, hyaluronic acid derivatives) and proteins (soy,
collagen, fibrin gels and silk) [11] and saturated poly-α-hydroxy esters, which include
poly(lactic acid) (PLA) and poly(glycolic acid) (PGA), and poly(lactic-co-glycolide)
(PLGA) for synthetic polymers [12]. Polymers can be very porous and biodegradable
but are problematic due to potential toxicity with degrading too quickly and poor material
strength.
Ceramics make a better cancellous replacement material to promote bone
regeneration. HA is a natural solution and material choice because of the bone’s
composition. HA nano-particles attract the cells to attach and form of extracellular
matrix within the porous tissue. However, HA does not work well individually because it
is brittle and fractures easily. Bioactive glass is also a ongoing researched material
because of its bioactivity and osteoconductivity. 45S5 Bioglass®, which is composed
on 45% , 24.5% , 24.5% and 6% in weight percent, is one of the
predominant bioactive glass compositions utilized.
Although neither a polymer nor a ceramic works well individually as a bone tissue
material for regenerative purposes, the composite of the different materials are being
42

43. proven as the best solution to fit all of the necessary attributes. The focus of this paper
was to compare and evaluate the porosity to the compressive strength of different
materials that incorporate nano-particles. Table 4 provides a summary of the porosity
and compressive strength of different materials currently under investigation. These
findings are also composed in Graph 9.
Compressive
Material Porosity (wt %) Strength (Mpa) Reference
Cancellous Bone 70 4 7
Cancellous Bone 90 0.2 7
Porous Diopside 75 1.36 8
Porous Diopside 90 0.2 8
45S5 Bioglass / nHAP 60 1 17
Shell Scaffolds 80 - 18
13-93 Bioglass 85 11 15
nHAP / PLA 86.2 0.44 16
nHAP / PLA 91.5 0.29 16
PLLA 87.5 1.8 21
nHAP / PLLA 85 8.5 21
HA / PLLA 86.5 4.6 21
nHAP needle / BCP / PCL 91 2.1 23
nHAP rod / BCP / PCL 91 0.9 23
nHAP sphere / BCP / PCL 91 1.4 23
BCP 94 0.1 23
HA / BCP /PCL - 0.55 23
nHAP 20% / PLGA 93 2.31 24
nHAP 10% / PLGA 91 1 24
nHAP 40% / PLGA 86 0.75 24
HA / PLGA 88 0.8 24
PLGA 87 1.1 24
BGC 10% / PLLA 92 0.34 25
BGC 20% / PLLA 91 0.33 25
BGC 30% / PLLA 88 0.35 25
PLLA 92 0.28 25
PLGA cylinder / PCL 30 9.22 28
PLGA tubular / PCL 35 9.8 28
PLGA tubular / PCL spiral 46 9.9 28
PLGA tubular / PCL nanofibre 44 9.1 28
Table 4: Summarized of various composites using nano particles and compares to micro particles.
43

44. 12
Porosity v. Compressive Strength
Cancellous Bone
Cancellous Bone
Porous Diopside
Porous Diopside
10 45S5 Bioglass / nHAP
13-93 Bioglass
nHAP / PLA
nHAP / PLA
Compressive Strength (MPa)
8 PLLA
nHAP / PLLA
HA / PLLA
nHAP needle / BCP / PCL
6 nHAP rod / BCP / PCL
nHAP sphere / BCP / PCL
BCP
nHAP 40% / PLGA
nHAP 20% / PLGA
4
nHAP 10% / PLGA
HA / PLGA
PLGA
BGC 10% / PLLA
2 BGC 20% / PLLA
BGC 30% / PLLA
PLLA
PLGA cylinder / PCL
0 PLGA tubular / PCL
0 20 40 60 80 100
Porosity (wt %)
Graph 9: Comparison of compressive strength against porosity of materials that incorporate nano-particles
Based on the data, utilization of nano-sized HA particle in a composite is better than
a micro-sized HA particle to create the necessary compressive strength of the
cancellous bone while still maintaining the porosity necessary to induce regeneration
and proper pathogen for minerals and other necessary body fluids. Beyond the use of
comparing porosity and compressive strength, the findings that reported bioactivity
found that materials incorporating nano-materials were more likely to find the necessary
osteoconductivity for cell formation leading to regeneration.
It is through the material selection that defines whether or not the material will serve
as a robust instrument to foster cell regeneration in the bone. Nano-materials are
proving themselves as a valuable asset in the body’s ability to regenerate. Through the
science of nanotechnology, regeneration of bone tissue will be possible for the body to
fully heal and repair itself without the need of prosthesis or substitution bone.
44

45. VIII. Bibliography
[1] L. Zhang, “Nanotechnology and nano-materials: Promises for improved tissue
regeneration,” Nano Today, Vol. 4, pp. 66 – 80, (2009).
[2] J. Venugopal et al., “Biomimetic hydroxyapatitie-containing composite nanofibrous
substrates for bone tissue engineering”, Philosophical transaction of the Royal Society,
Vol. 368, pp. 2065 – 2081 (2010).
[3] L. L. Hench, “Genetic design of bioactive glass”, Journal of the European Ceramic
Society, Vol. 29, pp. 1257 – 1265, (2009).
[4] M. Wang, “Composite Scaffolds for Bone Tissue Engineering”, American Journal of
Biochemistry and Biotechnology. Vol. 2, pp. 80-84, (2006).
[5] J.R. Jones, “New trends in bioactive scaffolds: The importance of nanostructure”,
Journal of the European Ceramic Society. Vol. 29, pp. 1275-1281, (2009).
[6] M. Stevens, “Biomaterials for bone tissue engineering”, Material Today, Vol. 11 nu. 5
(2008).
[7] M. Bohner, “Resorbable Biomaterials as bone graft substitutes”, Materials Today,
Vol. 13, nu. 1-2 (2010).
[8] C. Wu et al., “Porous diopside scaffold: A promising bioactive material for bone
tissue engineering”, Acta Biomaterialia. (2010), doi: 10.1016/j.actbio.2009.12.022.
[9] J. Jang et al., “Electrospun materials as potential platforms for bone tissue
engineering”, Advanced Drug Delivery Reviews, Vol. 61, pp. 1065 – 1083, (2009).
[10] M. Peter et al., “Novel biodegradable chitosan–gelatin/nano-bioactive glass ceramic
composite scaffolds for alveolar bone tissue engineering”, Chemical Engineering
Journal, Vol. 158, pp. 353 – 361, (2010).
45

46. [11] M. Swetha, et al., “Biocomposites containing natural polymers and hydroxyapatite
for bone tissue engineering”, International Journal of Biological Macromolecules, (2010),
doi: 10.1016/j.ijbiomac.2010.01.015.
[12] K. Rezwan, et al., “Biodegradable and bioactive porous polymer/inorganic
composite scaffolds for bone tissue engineering,” Biomaterials, Vol. 27, pp. 3413 –
3431, (2006).
[13] V.S.W. Chan, “Nanomedicine: An unresolved regulatory issue”, Regulatory
Toxicology and Pharmacology. Vol. 46, pp. 218-224, (2006).
[14] S. V. Dorozhkin, “ Bioceramics of calcium orthophosphates”, Biomaterials, Vol. 31,
pp. 1465 – 1485, (2010).
[15] Q. Fu et al., “Mechanical and in vitro performance of 13-93 biactive glass scaffolds
prepared by a polymer foam replication technique”, Acta Biomaterialia Vol. 4, pp. 1854 –
1864, (2008).
[16] C. R. Kothapalli, “Biodegradable HA-PLA 3-D porous scaffolds: Effect of nano-size
filler content on scaffold properties”, Acta Biomaterialia, Vol. 1, pp. 653 – 662, (2005).
[17] C. Vitale-Brovarone et al., “Development of glass-ceramic scaffolds for bone tissue
engineering: Characterisation, proliferation of human osteoblasts and nodule formation”,
Acta Biomaterialia Vol. 3 pp. 199-208 (2007).
[18] D. Bellucci, et al., “Shell Scaffolds: A new approach towards high strength
bioceramic scaffolds for bone regeneration”, Materials Letters, Vol. 64, pp. 203-206
(2010).
[19] O. Suzuki, “Octacalcium phosphate: Osteoconductivity and crystal chemistry”, Acta
Biomaterialia, (2010), doi: 10.1016/j.actbio.2010.04.002.
46

47. [20] X. Cai et al., “Preparation and characterization of homogeneous chitosan-polylactic
acid/hydroxyapatite nano-composite for bone tissue engineering and evaluation of its
mechanical properties”, Acta Biomaterialia, Vol. 5, pp. 2693 – 2703 (2009).
[21] E. Nejati et al., “Needle-like nano hydroxyapatite/poly(L-lactide acid) composite
scaffold for bone tissue engineering application”, Materials Science and Engineering C.
Vol. 29, pp. 942–949 (2009).
[22] Y. Wang, “Characterization of biodegradable and cytocompatible nano-
hydroxyapatite / polycaprolactone porous scaffolds in degradation in vitro”, Polymer
Degradation and Stability, Vol. 95, pp. 207 – 213, (2010).
[23] S. Roohani-Esfahani, “The influence hydroxyapatite nanoparticle shape and size on
the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite –
PCL composites”, Biomaterials, (2010), doi: 10.1016/j.biomaterials.2010.03.058.
[24] Y. Cui, et al., “The nano-composite scaffold of poly(lactide-co-glycolide) and
hydroxyapatite surface-grafted with L-lactic acid oligomer for bone repair”,Acta
Biomaterialia, Vol. 5, pp. 2680 – 2692 (2009).
[25] Z. Hong, “Preparation and in vitro characterization of scaffolds of poly (L-lactic acid)
containing bioactive glass ceramic nanoparticles”, Acta Biomaterialia Vol. 4, pp. 1297 –
1306, (2008).
[26] M. Peter, et al., “Nano-composite scaffolds of bioactive glass ceramic nanoparticles
disseminated chitosan matrix for tissue engineering applications”, Carbohydrate
Polymers, Vol. 79, pp. 284 – 289, (2010).
[27] A.M. El-Kady et al., “Development, characterization, and in vitro bioactivity studies
of sol-gel bioactive glass/poly(L-lactide) nano-composite scaffolds”, Material Science and
Engineering C, Vol. 30, pp. 120 – 131 (2010).
47

48. [28] J. Wang & X. Yu. “Preparation, characterization and in vitro analysis of novel
structured nanofibrous scaffolds for bone tissue engineering”. Acta Biomaterialia. (2010),
doi:10.1016/j.actbio.2010.01.045.
[29] Y. Liu, “Segmental bone regeneration using an rhBMP-2-loaded
gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model”, Biomaterials, Vol. 30, pp.
6276 – 6285 (2009).
[30] Jun-Hyeog Jang, et al., “Electrospun materials as potential platforms for bone
tissue engineering”, Advanced Drug Delvery Reviews, Vol. 61, pp. 1065 – 1083, (2009).
IX. Appendix
IX.I Fabrication methods available
There is a wide array of fabrication methods available and used for the different
materials available for bone tissue engineering. The choice of a fabrication method
directly impacts the material’s physical properties, including its porosity and mechanical
strength. The available fabrication methods all have strengths and weaknesses to its
use. Such strengths and weaknesses are outlined in the following table.
Fabrication Mode Advantages Disadvantages
Thermally induced phase High porosity (~95%), Long time to sublime solvent (48
separation highly interconnected pore structures, hours),
Anisotropic and tubular pores possible, shrinkage issues,
control of structure and pore size by small scale production,
varying preparation conditions use of organic solvents
Solvent casting / particle Controlled porosity, Structures generally isotropic,
leaching Controlled interconnectivity (if particles Use of organic solvents
are sintered)
Solid free-form Porous structure can be tailored to host Resolution needs to be improved
tissue, to the micro-scale,
48

49. Protein and cell encapsulation possible, Some methods use organic
Good interface with medical imaging solvents
Microsphere sintering Graded porosity structures possible, Interconnectivity is an issue,
Controlled porosity, Use of organic solvents
Can be fabricated into complex shapes
Sol-gel / Foam Sol-gel Easy to control Long time to create / multiple
steps to form
Electrospinning Control porosity, material strength Electric charge created in fibers
Easy machine set-up,
low cost of production
Table 5: 3-D composite scaffold fabrication methods and their advantages / disadvantages [7].
Research has been conducted, and is continued, to define how the different fabrication
techniques impact different materials based on the molecular composition of the
material utilized. Although it was outside the scope of this paper to analyze the different
forms of fabrication, a brief outline of the preferred (or utilized) method of fabrication for
different nano-materials can be seen in the following table.
Name Method Produced
Polyhydroxybutyrate (PHB) and polyhydroxybutyrate- Electrospinning, selective laser
co-valerate (PHBV) sintering
Polycaprolactone – tricalcium phosphate (PCL-TCP) Electrospinning
Polycaprolactone (PCL) and nano-hydroxyapatite Melt-molding / leaching
(nHA)
Poly-L-lactides (PLLA) and bioactive glass ceramics Sol-gel
Calcium phosphate cement (CPC) – tetracalcium Melting - mixing
phosphate and dicalcium phosphate anhydrous
Tricalcium phosphate (TCP) and hydroxapatite (HA) Sol-gel
Polycaprolactone (PCL) and polylactide-coglycolide Melt-molding / porogen leaching,
(PLGA) electrospinning
Nano-hydroxyapatite (nHA) and poly-L-lactides (PLLA) Solid-liquid, wet chemical, thermally
induced phase separation
Table 6: Currently researched Nano-materials used for bioactive scaffolds in Bone Regeneration
49

50. IX.II Nano-fibres
Nano-fibres is a broad type of nano-material produced in a particular fashion by its
fabrication process. Nanfibres are typically created through electrospinning. There is a
great deal of research already conducted on this topic. This form of material formation
was lightly reviewed in this report because of the current trend of novel research
focused on nanoparticles rather than nano-fibres. Nevertheless, a table is attached
which shows the wide variety of materials that can be spun into nanfibres for use as
bioactive scaffolds. [30].
Figure 14: Summary of Electrospun nanfibre materials for bone reconstruction [30].
50