Bioactive Scaffolds for Bone TissueRegeneration: Emerging Nano-materials             Final Report              Prepared by...
Outline   Bioactive scaffold material is becoming more important and is on the cutting edge ofresearch today because of th...
I.     IntroductionI.I.        General Overview       The medical sciences continue to advance in parallel with improvemen...
To regenerate bone tissue, the body relies on scaffolds. Scaffolds are a type oftemplate the body uses to regenerate tissu...
scale. The cancellous bone has been measured to have a compressive strength of4000 kPa when 70% porous and still maintain ...
international (SI) units. Therefore, stress is measured in Pascals or Newtons per metersquared. [http://www.engineersedge....
Figure 1: Snapshot of different nano-materials available for bone tissue regeneration (A) Scanningelectron microscopy (SEM...
fundamental reason why nano-materials are important in bone tissue regeneration isbased, primarily, in a bottom-up approac...
tropocollagen macromolecules, which self-assemble into type I collagen, thepredominant collagnous molecule present in the ...
Fig. 2. Schematic of osteogenic progenitor cell cycle leading to (1) apoptosis; (2) mitosis and cellproliferation; or (3) ...
bone, the ECM plays a viable role and any material used in regeneration process  should assist the cells in the matrix. Si...
promote greater amounts of specific protein interactions to more efficiently stimulatenew bone growth compared to conventi...
natural bone. Studies of polymer, ceramic, and composite materials will now bereviewed to demonstrate why nano-materials a...
weights and copolymers. However, these polymer-based scaffolds can also failprematurely because of a bulk erosion process ...
III.III     Risk associated with Polymers   One of risk with using a synthetic polymer is its potential toxicity. While ce...
apatite layer on the surface of a bioactive ceramic can be reproduced in a protein-freeand SBF, which has an ion concentra...
Fig. 5. SEM images of nHAP particles in form of(A) needle, (B) spherical and (C) rod shaped [16].IV.II. Bioactive Glass   ...
Na ions, depending on the processing route and particle size. Furthermore, the rate ofbioresoprtion of bioactive glasses c...
method, the slurry used was prepared by dispersing the glass powders into distilledwater together with a polyvinylic binde...
suggest that the fabricated 13–93 glass scaffolds could be applied as biologicalscaffolds for repair and regeneration.   F...
but weaker fracture toughness. Therefore, HA and calcium phosphates cannot be usedalone for load-bearing scaffolds but rat...
mechanical strength was evident after 14 days of soaking. Overall, the research hereshows that diopside scaffolds possess ...
and size, freeze-drying parameters etc play important roles in forming the desiredcomposite scaffold porous structure. A v...
An early study, published in 2005, created a composite composed of nano-hydroxyapatite (nHAP) and poly(lactic acid) (PLA) ...
scaffold material and demonstrates osteoconductivity, biodegradability, and mechanicalstrength that is comparable to cance...
Another study, carried out by E. Nejati et al., combined nHAP to PLLA because ofthe superior mechanical strength that is f...
The use of nano-hydroxyapatite (nHAP) has also been paired with polycaprolactone(PCL) in a study carried out by Y. Wang et...
water infiltration. Both types of materials displayed relatively stable mechanicalproperties and stable pore interconnecti...
Graph 4. Compressive strength of samples: BCP, BCP with micro HA coating, BCP with PCL coating,BCP with PCL/nHAP needle co...
Graph 5. Osteoblast proliferation analysis [24].    HA and nHAP are a logical material of choice in bone tissue regenerati...
A few reported studies using nano-bioactive glass ceramic particles (nBGC) wereconducted by M. Peter et al. Both of these ...
Fig. 9. SEM micrograph showing the macroporous microstructure of CG (a and b) and composite scaffold(c and d). Pore size r...
Fig. 10. SEM morphology for the porous PLLA/BGC scaffolds with different BGC contents: at low-magnification: (A) 0 wt.%, (...
PLLA/BGC composites increased with the introduction of BGC nanoparticles, whichhave a hydrophilic character. A mass increa...
of the material was shown to increase with both the time soaking in SBF and with theincrease of the glass content.   Resul...
characteristics regarding porosity and mechanical strength which put the design andmaterial composite as good candidates f...
Composite materials have been heavy reviewed recently for bone tissueregeneration because of the ability to balance the st...
emerged at 4 weeks post-surgery in the HA/PLGA, 10 and 20 wt% nHAP/PLGA and byweek 24, all samples containing HA or nHAP w...
Fig. 14. Typical radiographs of radius resection implanted with composites: untreated control (A1&2),PLGA (B1&2), 5 wt.% o...
first material or second material mentioned above or nothing. The animals underwentX-ray examinations at 4, 8, and 12 week...
Fig. 15. Radiographs of a rabbit radial bone defect repaired with different scaffolds at 4, 8 and 12 weeks.(A–C) No graft ...
requirements for clinical use. Research has been conducted on polymers, ceramicsand composites. A number of different fabr...
proven as the best solution to fit all of the necessary attributes. The focus of this paperwas to compare and evaluate the...
12                                                         Porosity v. Compressive Strength                               ...
VIII. Bibliography[1] L. Zhang, “Nanotechnology and nano-materials: Promises for improved tissueregeneration,” Nano Today,...
[11] M. Swetha, et al., “Biocomposites containing natural polymers and hydroxyapatitefor bone tissue engineering”, Interna...
[20] X. Cai et al., “Preparation and characterization of homogeneous chitosan-polylacticacid/hydroxyapatite nano-composite...
[28] J. Wang & X. Yu. “Preparation, characterization and in vitro analysis of novelstructured nanofibrous scaffolds for bo...
Protein and cell encapsulation possible,      Some methods use organic                            Good interface with medi...
IX.II   Nano-fibresNano-fibres is a broad type of nano-material produced in a particular fashion by itsfabrication process...
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Bioactive Nanoparticle Materials for Bone Tissue Regeneration (BioE 505 -- NanoBioTechnology)

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  1. 1. Bioactive Scaffolds for Bone TissueRegeneration: Emerging Nano-materials Final Report Prepared by: Kathleen Broughton NanoBioTechnology BIOE 505 April 13, 2010
  2. 2. Outline Bioactive scaffold material is becoming more important and is on the cutting edge ofresearch today because of the foreseeable need for bone tissue regeneration as aneffective way to improve the current medical practice of bone replacement. Boneregeneration is more effective because there are no concerns of the body rejecting theprosthesis, less concern regarding contamination in a surgery leading to infection aswell as less patient rehabilitation. To design a good bioactive scaffold there are a number of considerations that mustbe taken into account. There has been a good deal of research at the micro-scale levelon this subject and research is now gearing itself to study nano-materials, which will bevaluable 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 bestmaterial available to assist in bone tissue regeneration. The paper will first discuss theneed for nano-materials and provide background information with respect to boneformation. The discussion will then shift to background information about polymer andceramic materials studied. This foundation will allow for an informative discussion ofceramic materials, which is where the bulk of nano-material research is conductedtoday. The main comparison made in this paper is focused on comparing the porosityto the mechanical (compressive) strength of different materials. A brief analysis of an invivo 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 opportunityto aid in the development of this research. 2
  3. 3. I. IntroductionI.I. General Overview The medical sciences continue to advance in parallel with improvements to the careand methods to treat an aging population because of technological innovation. One ofthe ever growing needs for our aging society is that of bone replacement andregeneration. Tissue begins to decay progressively in humans starting around agethirty. The National Center for Health Statistics (NCHS) reported 1,039,000 bonefractures for all sites in 2004 in the U.S alone. Additionally, around 118,700 patients hadosteoarthritis and associated disorders in 2000. The American Academy of OrthopedicSurgeons reported that in just a 4 year period, there was an 83.72% increase in thenumber of hip replacements performed from nearly 258,000 procedures in 2000 to474,000 procedures in 2004 [1]. The orthopedic market was estimated to be worthapproximately US $37.1 billion in 2008, following a growth of 9.7 percent over theprevious year [2]. Furthermore, the development of sturdy prostheses has not advancedbeyond the medical instrumentation to maintain life. In other words, patients are nowoutliving their replacement parts; studies have shown that there is a 15 – 50% failurerate to prostheses over a 15 – 30 year life span [3]. These statistics have driven themedical community to research bone tissue regeneration as an alternative and betterway of replacing bone tissue loss. Medical technology continues to make great strides in tissue replacement throughallographs (donor replacement) and autographs (self-donor replacement).Replacement of aged, diseased or damaged tissues is more common today because ofreliable and affordable biomaterials and the perfection of surgical procedures forprostheses implantation and subsequent rehabilitation. However, bone replacement issusceptible to prosthesis implant rejection, transmission of diseases associated with thetransplant, shortage of available donors, continued physical therapy, and a highfinancial investment compared to regeneration [4]. Therefore, regenerative bone tissueimplantation is a beneficial alternative to replacing the bone tissue through prosthesis. 3
  4. 4. To regenerate bone tissue, the body relies on scaffolds. Scaffolds are a type oftemplate the body uses to regenerate tissue, particularly bone tissue. The body is verygood at healing and regenerating itself when the defects are small. However, the bodycannot heal larger defects without the use of an aid. There are multiple criteria for boneregeneration scaffolds which include: bioactivity (ability to bond to bone), osteogenic(stimulation of bone growth), biocompatible (induce minimal toxic or immune responsein 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, andmeet all regulatory requirements for clinical use [5]. In terms of the osteogenicmaterials, the material should be osteoinductive (capable of promoting thedifferentiation of progenitor cells down an osteoblastic lineage), osteoconductive(support bone growth and encourage the ingrowth of surrounding bone), andosteointegrate (integrative to the surrounding bone) [6]. Since bone tissue is the mostcommonly replaced organ of the body, the necessity to engineer the proper scaffoldmaterial is of a growing need. Over the past decade, great strides have taken place in medical science to find anadequate material to serve substitutionally and promote regeneration. Most scaffoldmaterials 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 havetaken part from both synthesis and material perspectives in aid of discovering a materialthat fits all parameters of natural bone. The most difficult parameters to balance are themechanical strength of the material under a compression load against the porosity andthe resorbability of the scaffold material.I.II Method of Analysis Cancellous bone, the spongy internal bone with an open porous network, naturallyhas strong compressive strength and simultaneously good porosity for nutrientabsorption. Approximately 70% of natural bone is composed of hydroxyapatite particles( (HA) and the remaining 30% is an organic matrix, mainly collagen typeI. The structure of the bone is hierarchically organized from a macro to micro to nano- 4
  5. 5. scale. The cancellous bone has been measured to have a compressive strength of4000 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 tosynthesize. The resorbability of the filler material is controlled through two differentmeans [8]. In the first approach, the geometry of the material is optimized; In otherwords, the pores are balanced against the compressive strength. In the secondapproach, the chemistry is modified in the material choice. The manner in which amaterial is fabricated impacts its performance as a biomaterial and, as such, the focusof this paper is a comparison of different materials (nano and micro) in terms of porosityand mechanical strength. [author note: there is a wide array of fabrication methodsused for scaffold material; such discussion is not of primary focus of this report. A briefdiscussion of the fabrication methods with the strengths and weaknesses of suchmethods along with an outline of the different material’s optimized fabrication method isavailable in the appendix.] To calculate the porosity of a scaffold, a researcher could take a variety of differentformulas to reach the same conclusion. A general formula is based on weightmeasurement according to: Where is the total porosity content (% vol.), is the measured weight of thescaffold and is the theoretical one obtained by multiplying the scaffold volume andthe material density [8]. To calculate the compressive strength of a material, a general formula should beapplicable based on the equation: Where σ is the stress (known as compressive strength of a material when the forceapplied is perpendicular to the stress plane), F is the force penetrated into the materialand A is the surface area of the material that was impacted by the force The applied unitsystem in bone tissue engineering research is consistent with utilization of standard 5
  6. 6. international (SI) units. Therefore, stress is measured in Pascals or Newtons per metersquared. [http://www.engineersedge.com/material_science/stress_definition.htm]. An additional formula, not heavily analyzed in the thesis because it is notconsistently reported like porosity and compressive strength, is based on thedegradation of the material and is based upon the formula: Where is the initial weight of the sample, and is the dry weight of the sampleafter 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 willserve 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 aform, which independently or with the interaction of the system, is used to direct, bycontrol of interactions with components of living systems, the course of any therapeuticor diagnostic procedure, in human or veterinary medicine [8]. In other words,biomaterials are intended to interface with biological systems to augment, repair, orreplace any tissue, organ or function in the body. The key characteristic of biomaterialsis its ability to remain in the biological environment without damaging its surroundings oritself. 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 previousresearch conducted with materials that utilize nano-particles, with a particular focus onthe porosity and mechanical strength of the various materials and (3) hypothesizingwhat 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. 7. Figure 1: Snapshot of different nano-materials available for bone tissue regeneration (A) Scanningelectron microscopy (SEM) image of poly(L-lactic acid) (PLLA) nanofibrous scaffold with interconnectedspherical macropores created by a phase-separation technique. (B) Electrospun polycaprolactone/hydroxyapatite/gelatin (PCL/HA/gelatin, 1:1:2) nanfibres which significantly improved osteoblast functionsfor 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 electronmicroscopy (TEM) image of monodispersed magnetic Fe3O4 nanoparticles (6 nm) deposited from theirhexane dispersion and dried at room temperature. [11]. II. Need for Nano-based Materials Research for scaffold materials that cause regeneration in bone has become moreaggressive in the last few years (months!). The need to investigate scaffold material atthe nano level is necessary because the previous attempts, at the micro level, havebeen proven unfruitful to find a material that possesses the same characteristics ofnatural bone. Nanotechnology has made significant advances over microtechnology inthis field of research. Nano-materials, which are materials with basic structural units,grains, particles, fibers, or other constituent components smaller than 100nm in at leastone dimension, have become more prevalent in research the last few years. The 7
  8. 8. fundamental reason why nano-materials are important in bone tissue regeneration isbased, 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 HAnanocrystallites and collagen fibrils are organized. The main way to produce artificialbiomaterials 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 inregeneration. To understand the material selection, it is important to understand thefundamentals of bone regeneration and how bone develops. The fundamental thought of using materials for bone regeneration rather than bonereplacement with bioactive bonding was stated by a J. Wilson et al. in 1992 afterreporting that new bone had colonized on the surface of a monkey jaw while usingbioactive glass to replace periodontal-diseased bone [3]. This phenomenon waslabeled as osteoproduction and was soon seen by other researchers with similarmaterials. A second key in developing the base of bone regeneration was the finding ofXynos et al. that the ionic dissolution products released from the bioactive materialinfluenced the osteogenic precursor of the cells and the cell reproduction. This elementin the development focused on the slow degregation rate of the material and the foundSi and Ca ions that were slowly released from the material during osteostimulation. Thethird key in developing the base of bone tissue regeneration is focused on theconcentrations of the ionic dissolution products that activate or up-regulate certainfamilies of genes in osteogenic cells. These studies were confirmed by a collaborationof L.L. Hench, the founder of discovering the bioactivity of certain glass, and ProfessorDame Julia Polak. The general process of osteoprogenitor cell reproduction is similar to all types of cellreproduction. As shown below the cell completes multiple phases for bone regeneration[3]. The first step is labeled G1 where the osteoblasts are synthesizing phenotypicspecific cellular products. A full functioning osteoblast produces osteocalcin and 8
  9. 9. tropocollagen macromolecules, which self-assemble into type I collagen, thepredominant collagnous molecule present in the bone matrix and numerous otherextracellular matrix proteins. Osteocalcin is a bone extracellular maxtrix non-collagenous protein produced by mature osteoblasts with a synthesis that correlateswith the onset of mineralization, a critical feature of new bone formation. Following theG1 cycle, the cell enters the S phase where DNA and RNA synthesize. Once thesequencing 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/RNAsequencing. If the chemical environment of the cell is not acceptable the cell will gothrough apoptosis, die. If the cell passes its self-programmed test to specifications itgoes through mitosis, the M phase, and forms two descendant osteoblast cells. Thisprocess is then repeated to form new generational cell replications. The self-regulationof 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 regenerationaround the age of thirty. For bone regeneration to fully occur, the osteoblast cell mustundergo differentiation and form into an osteocyte, which are not capable of celldivision. Osteocytes represent the cell population responsible for extracellular matrixproduction and mineralization, the final and most critical step in bone development.Understanding the regeneration of osteoblast and osteocyte cells is valuable todetermine 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 aretype 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 differentiationcycle mentioned above. Osteoblasts that form into osteocytes are responsible forintercellular communication within the bone and also to break down the bone matrix andrelease calcium. Candaliculi provides the opportunity for nutrients and oxygen to passbetween the blood vessels and the osteocytes. Osteoclasts are polarized cells andrequire mineral dissolution followed by degradation to be reabsorbed into the bone. 9
  10. 10. Fig. 2. Schematic of osteogenic progenitor cell cycle leading to (1) apoptosis; (2) mitosis and cellproliferation; 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. 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. 12. promote greater amounts of specific protein interactions to more efficiently stimulatenew bone growth compared to conventional materials. Nano-material based scaffoldscan be grown or self-assembled through processes such as electrospinning, phaseseparation, self-assembly processes, sintering, and solvent casting / leaching. Tofabricate 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 hierarchalself-assembly of bone. (B) Nanophase titanium (top, the atomic force microscopy image) andnanocrystalline HA/HRN hydrogel scaffold (bottom, the SEM image). (C) Schematic illustration of themechanism 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 ofprotein 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. 13. natural bone. Studies of polymer, ceramic, and composite materials will now bereviewed to demonstrate why nano-materials are necessary to successfully regeneratebone tissue. III. Polymer Based MaterialsIII.I. Natural-based Polymers There are two types of natural biodegradable polymers; polysaccharides (starch,alginate, chitin/chitosan, hyaluronic acid derivatives) and proteins (soy, collagen, fibringels and silk) [11]. Biofibers such as ligneocellulosic natural fibers are also used asreinforcements to these polymers. Chitosan, for example, is a good polymer for scaffold regeneration because itdegrades at a rate similar to that of new tissue formation and does not release toxinsduring degradation leading to complications, such as inflammation. Chitosan has beenfound to work well with natural bone and has excellent osteoconductivity andbiocompatibility. Thus, chitosan has been researched in conjunction withhydroxyapatite (HA) and has been found to have good mechanical properties as acomposite. Further discussion of chitosan in composites is discussed below.III.II. Synthetic-based Polymers Synthetic polymers can be produced under controlled conditions and, therefore, arequite predictable and reproducible in terms of mechanical and physical properties, suchas tensile strength, elastic modulus and degradation. In terms of polymers, the most often utilized biodegradable synthetic polymers aresaturated poly-α-hydroxy esters, which include poly(lactic acid) (PLA) and poly(glycolicacid) (PGA), and poly(lactic-co-glycolide) (PLGA) [12]. The chemical properties ofthese polymers allow hydrolytic degradation through de-esterification. Upondegradation, the monomeric components are removed naturally by the body through itspathways. PLA and PGA can be processed easily and the degradation rates, physicaland mechanical properties are adjustable over a wide range by using various molecular 13
  14. 14. weights and copolymers. However, these polymer-based scaffolds can also failprematurely because of a bulk erosion process or cause a strong inflammatory flairthrough 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 molecularcomposition of the material. PLGA, for example, has a wide range of degradation ratesgoverned by both hydrophobic/hdydrophilic balance and crystallinity all which is basedupon the composition of the molecular chains of the material. The degradation of thepolymers mentioned thus far occurs in the bulk of the material; some polymers degradeon the surface. Surface bioeroding polymers undergo a heterogeneous hydrolysis process and thuserode 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 degradablematerial 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 materialand is discussed further below. 14
  15. 15. III.III Risk associated with Polymers One of risk with using a synthetic polymer is its potential toxicity. While ceramicbased biodegradable materials are found within bone, polymer material is not. Thisconcern, of toxicity, is present not only in polymer material, generally, but also withnano-materials. Nanotechnology research is considered too recent for any true andaccurate studies to be completed to determine the different rates of toxicology based onnanoparticles [13]. It was suggested that nanoparticles, because of their small sizes,could act in a manner which would modify protein structures, either by altering theirfunction or rendering them antigenic, thus raising their potential for autoimmune effects.Concerns of nano-materials have been raised by both the National Science Foundationand the Environmental Protection Agency. Currently, there is no specific regulatoryrequirement to test nanoparticles. It does, however, remain heavily on the radar ofregulators. Until an understanding of the actual toxicity of nano-materials is determined,however, research will continue to stride in this direction because the benefits tomedical science and society are greater than regulating the unknown. IV. Ceramic Based Materials Ceramics are well known and used for scaffold fabrication in bone tissueengineering and are well studied because of their similar characteristics to natural bone.As previously mentioned, cancellous bone is approximately 70% HA nanoparticles. It iseasily fitting, therefore, that a replacement material to spark regeneration is composedof a ceramic based material. Bioceramic materials are organized into three classes:relatively bioinert ceramics, bioactive (or surface reactive) and bioresorbable [14]. Thereactivity of bioactive ceramics in simulated body fluid (SBF) demonstrates the ability ofthe material to react well to ions. A characteristic of bioactive glass and similar ceramicmaterials, for example, is a kinetic surface modification that occurs to the material’ssurface upon implantation. The surface forms a biologically active hydroxyl carbonateapatite (HCA) layer, which provides the bonding interface with tissues. The HCA phasethat forms on the bioactive implant is chemically and structurally equivalent to themineral phase in bone and provides interfacial bonding. The in vivo formation of an 15
  16. 16. apatite layer on the surface of a bioactive ceramic can be reproduced in a protein-freeand 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 andbioactive glass. New materials under study, such as octacalcium phosphate are alsobriefly 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, morefocus is on the later material because of its crystallographic properties, biocompatibility,bioactivity and osteoconductivity [2]. Studies have been conducted in pairing nHAP toboth natural polymers and synthetic polymers. The use of nHAP is predominately donein the form of particles or fibres and is increasing in use for a composite materialbecause of the improved physical, biological and mechanical properties of the scaffoldmaterial when incorporating the nHAP. Studies conducted with the incorporation ofnHAP are discussed in the ceramics section below. 16
  17. 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 wellas osteoconductivity was made by Larry Hench et al. in 1969. Bioactive glass developsa calcium-deficient, carbonated phosphate surface layer that allows it to chemicallybond to bone. This bioactivity is associated with the formation of a carbonated HA layeron the glass surface when implanted or in contact with biological fluids [3]. Thecapability of a material to form a biological interface with the surrounding tissue iscritical to bone regeneration. The fact that bioactive glass also promotes regenerationand is biodegradable makes it a prime candidate as a bone regeneration material. One of the predominant materials is 45S5 Bioglass®, which is composed on45% , 24.5% , 24.5% and 6% in weight percent. It has been foundthat bioactive glass surfaces can release critical concentrations of soluble Si, Ca, P and 17
  18. 18. Na ions, depending on the processing route and particle size. Furthermore, the rate ofbioresoprtion of bioactive glasses can be controlled through modifying the chemicalproperties of the material. [12]. A primary disadvantage of bioactive glass, however, isthat when the material crystallizes the bioactivity significantly decreases to the extent ofbecoming an inert material. Crystallization of bioactive glass occurs by viscous flowsintering. Sintering is necessary to create dense scaffold struts that hold mechanicalstability to that of natural bone. Another drawback of bioactive glass is its low fracturetoughness and mechanical strength which provide for poor load-bearing situations.Nevertheless, bioactive glass is a researched material because it has many favorableattributes. In an effort by C. Vitale-Brovarone et al., characterization research was conducted infurther developing the 45S5 bioactive glass [17]. Their team determined that porestructure 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 spongeimpregnation technique to prepare the scaffold. In the manner of preparation, polymericsponges possessing an open, trabecular structure are used as a template for a ceramicreplica through impregnating the sponge with a slurry mix of ceramic powders and thenprocess the materials with heat. Results show the pore strut thickness from 5-20microns and the trabecular structure closely resembles that of natural bone. Thecompressive strength of about 1 ± .4 MPa was found in the samples. In the SBF, thescaffold material surface was covered by a silica-rich layer of HA crystals. Calciumdeposits were also discovered on the scaffolds. These results add to a foundation forfurther research in bioactive glass. Another team of researchers also utilized 45S5 Bioglass® as a foundation forresearching scaffold replication and created shell scaffolds. [18]. Here, the focus is onforming a material with high porosity and adequate mechanical properties. Althoughmost bioactive glass scaffolds are created by the foam replication method, this teamfocused 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 butdoes not change the porosity that is currently similar to that of cancellous bone. In this 18
  19. 19. method, the slurry used was prepared by dispersing the glass powders into distilledwater together with a polyvinylic binder. The optimized weight ratio of the componentswas: 59% water, 29% 45S5 Bioglass®, and 12% polyvinylic binder. The aim of thecompositions was to increase the final porosity of the samples, in particular at thesurface. At the end of the process of burning-out the samples, the sponges resulted tobe coated by a thin shell made of bioactive glass and binder. The process of outer shellcoating with slurry and drying was repeated to obtain a thicker outer shell. The scaffolds produced have an original structure with an internal structurecharacterized by high porosity and thin walls with an external resistant surface similar toa shell with strong mechanical properties. The shell scaffold can be easily handledwithout damage and the porosity is sufficient to ensure excellent permeability to cellsand fluids. For the samples under review, the average total porosity calculated is about80% vol. This value satisfies the requirements to allow bone tissue in-growth. Thisteam plans to follow-up with further investigation of the mechanical strength of thematerial as well as its reaction to immersion in simulated body fluid. With time, researchers have scaled down the size of the bioglass particles. One ofthe studies leading to the use of nano-materials with bioactive glass was performed byQ. Fu et al. [15]. In their study, a polymer foam replication technique was used toprepare porous scaffolds of 13–93 bioactive glass. The 13-93 glass is approved for invivo 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–staincurve was 3.0 ± 0.5 GPa. Taking the compressive strength as the highest stress on thestress–strain curve, the average compressive strength was 11 ± 1 MPa for eightsamples tested (porosity = 85 ± 2%). SEM images of the surface of a 13–93 glassscaffold were taken after immersion in an SBF for 7 days. Compared to the smoothglass surface of the as-fabricated construct, the treatment in the SBF produced a fineparticulate surface layer. High-resolution SEM images showed that the surfaceconsisted of a porous network of nanometer-sized, needle-like crystals. The results 19
  20. 20. suggest that the fabricated 13–93 glass scaffolds could be applied as biologicalscaffolds 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, arecurrently under study as a filler or coating to form the ECM. Calcium phosphates, HAand tricalcium phosphate, has also been researched as a possible ceramic basedscaffold material. Calcium phosphates have excellent biocompatibility due to their closechemical and crystal resemblance to bone mineral [14]. Calcium phosphates possessosteoconductive properties and can bind bone directly under certain conditions. Thedownside of HA and calcium phosphates is their slow biodegradation rate and lowmechanical strength under load-bearing stress. The dissolution of synthetic HAdepends on the type and concentration of the buffered / unbuffered solutions, pH of thesolution, degree of the saturation of the solution, and the composition and crystallinity ofthe HA phase. Crystalline HA exhibits the slowest degradation rate in comparison toother calcium phosphates. HA, compared to bone, has a better compressive strength 20
  21. 21. but weaker fracture toughness. Therefore, HA and calcium phosphates cannot be usedalone for load-bearing scaffolds but rather work well in a composite material. A modified version of calcium phosphate recently under review is octacalciumphosphate (OCP). It structure is similar to HA and shows better osteoblastic activity invivo when compared to calcium phosphate. OCP may stimulate osteoconductivitybecause 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 progressiveimplantation periods. One of the set-backs of calcium orthophosphate is its highbrittleness, 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 diopsidebecause of a need to find a material with adequate mechanical strength in load-bearingsituations. 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 wereprepared using the polymer sponge template method where the foam template, with a25 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 slurryinto the foam. The sponge was then dried at 60 degrees Celsius for one day andsintered at 1300 degrees Celsius for three hours. The phase composition, poremorphology and microstructure of the sintered scaffolds were characterized by X-raydiffraction and scanning electron microscopy. The results of this study show that show a uniform inner network structure and theinterconnectivity of the scaffolds was approximately 97% complete. Results also showthat with the increase of porosity from 75% to 90%, the compressive strength andcompressive modulus of diopside scaffolds decreased from 1360±370kPa and 68±20MPa to 200±20KPa and 10±3.3MPa, respectively. The diopside scaffolds show stablemechanical properties while soaking in SBF up to 14 days, but the weakened 21
  22. 22. mechanical strength was evident after 14 days of soaking. Overall, the research hereshows that diopside scaffolds possess enhanced mechanical strength and mechanicalstability and decreased degradation rate compared to bioglass and scaffolds,which leads to their conclusion that diopside scaffolds could be a promising candidatefor bone tissue regeneration. Although ceramics are more heavily studied as an appropriate bioactive scaffoldmaterial for bone tissue regeneration, composite materials with ceramic are morepromising to balance all of the requisite characteristics of scaffolds. Furthermore, nano-materials are more prevalently under investigation currently in composite materialsbecause 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 desirablequalities of the chosen materials. By utilizing a composite for a scaffold material themechanical strength and bioactivity of a ceramic can be paired with the porosity anddegradation behavior of a polymer. Inclusion of bioactive glasses has been shown tomodify surface and bulk properties of composite scaffolds by increasing thehydrophilicity and water absorption of the hydrophobilc polymer. Ideally, thedegradation and resorption of composite scaffolds are designed to allow cells toproliferate and secrete their own extracellular matrix while the scaffolds graduallydisintegrate. There are a multitude of fabrication methods available to create compositescaffolds, which are outlined in the table 1, but which discussion is beyond the scope ofthis paper. The research of composite material for bone tissue regeneration, especiallywith use of nano-materials, is currently in their incarnation stages. Composite materials can offer the best qualities of each material when properlybalanced and fabricated. There are two approaches used to make a bioceramic-polymer composite scaffold: incorporating bioceramic particles in the scaffold through avariety of techniques and coating a polymer scaffold with a thin layer of apatite throughbiomimetic processes. Factors such as polymer solution concentration, porogen type 22
  23. 23. and size, freeze-drying parameters etc play important roles in forming the desiredcomposite scaffold porous structure. A variety of materials have been researched insearch 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 tissueregeneration because of the materials osteoconductive characteristic since the maincomposition of natural bone tissue is HA particles. Since the cancellous bone materialis approximately 70% nHAP, it is intuitive to make a scaffold from this material. Asshown through the following examples, the use of a nano-scale HA particle showsbetter results in terms of osteoconductivity, compressive strength and bioactivity incomparison to micro-scale HA particles used in a composite material. 23
  24. 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 sizewas 25 nm by 150 nm. In this early study, the researchers recognized that the nHAPserves as a better filler material than micro-HAP because it has more homogenousdistribution in the scaffold, which directly affects the mechanical properties of thematerials. The finding of the study indicated that the nHAP particles formed varied insize between 25 and 50 nm length by 150 – 300 nm length and were phatelet-shapedwith a uniform morphology. Fig. 7. (a) TEM images of the nHAP particles; (b) selected area electron diffraction pattern of the HAparticles shown in (a) [16]. Further results showed that the scaffolds swelled from water absorption becausePLA is highly hydrophobic. The scaffolds, when immersed in water for 24 hours had anincrease of 540% for pure PLA and 274% when 50wt% of the scaffold was nHAP. Theporosity 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. Themechanical 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 involvingnHAP because the overall conclusion was that nHAP provides a solid structure for the 24
  25. 25. scaffold material and demonstrates osteoconductivity, biodegradability, and mechanicalstrength that is comparable to cancellous bone. A more recent study of nHAP with PLA was conducted by Cai et al. when the studyincorporated nHAP with chitosan in the presence of PLA [20]. The choice of chitosanwith nHAP was natural because of the chemical composition of bone. These materialstogether show strong biocompatibility and also has strong regenerative efficacy andosteoconductivity. PLA was introduced because it has a high mechanical strength andis widely used for implants. In this study, nHAP rod-shaped particles that wereapproximately 50 nm by 300 nm were homogeneously distributed into a chitosan/PLAmatrix and studied in terms of morphology and mechanical properties. SEM results indicate that the nHAP particles have a tendency to disperse in thechitosan, which indicates that the PLA was directing the interaction of the nHAP.Observations also indicate that the PLA nanoparticles, which varied in size between 30to 50 nm, were heterogeneous and rationalize why the nHAP was induced to goodosteoconductivity. This heterogeneous nucleation was also caused by the mineralcrystallinization of the calcium and phosphate ions in the materials and the boneregenerating. The compressive strength of the chitosan/nHAP with different HAcontent, ranging from 50-80%, was measured with test samples including and notincluding PLA. The porosity was not measured in this study. Results indicate that a 70wt.% nHAP content held the best compressive strength for the two sample types (withor 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 polymersincrease the bioactivity and osteoconductivity of the composite scaffold compared tomicro-sized particles or no particles. The study conclusion also found the closecombination between the nano-sized inorganic particles and the organic matricesenhancing the mechanical properties with still providing the proper shape and size fortissue growth. The third conclusion made in this study was that nHAP can acceleratethe formation of bone tissue apatite in comparison to micro or no HA particle. 25
  26. 26. Another study, carried out by E. Nejati et al., combined nHAP to PLLA because ofthe superior mechanical strength that is found in the composite [21]. Since natural boneis comprised of HAP nanocrystallites, it is natural to use this material in a syntheticcomposite for bone regeneration. The choice of PLLA was chosen because of its lowtoxicity, good mechanical characteristics, and its predictable degradation rates. Theanalysis 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 nHAPparticles were distributed within the porewalls of the nano-composite scaffold and noaggregation appeared in the pores. The pore size of pure PLLA and nano-composite scaffolds were in the range of 167to 95 μm, respectively. The mechanical properties indicate the average of bothcompressive modulus and strength of the microcomposite (13.68 and 4.61 MPa) andthe nano-composite (14.07 and 8.46 MPa) scaffolds are statistically significant higherthan those of pure PLLA scaffolds (1.79 and 2.4 MPa). Furthermore, the compressivestrength of nanocomomposite HAP is greater than that of micro HAP when evaluatedwith 50% weight percent HAP. These findings validate that nano-composites make abetter 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. 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 basedon its biodegradability, good biocompatibility and excellent mechanical strength. Asmentioned, nHAP is representative of composition and structure similar to natural boneand has good biocompatibility, osteoconductivity and osteoinductivity. The compositematerial was fabricated by a melt-molding / porogen leaching technique using poly(ethylene glycol) and NaCl particulate. The analysis focused on degradation after sixmonths of in vitro sublimation in a plasma blood solution and included review of theweight loss, nHAP content, PCL molecular weight, morphology, and mechanicalproperties of the material.Fig. 8. SEM photographs of the MSCs cultured on the top surface of the PCL scaffold and the nHA/PCL-1scaffold. Arrows show the spherical cell after day 1 (a, c) and the cell–cell contacts with fibrousextracellular 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 compositescaffold to the decrease in the nHAP loss. Degradation of nHAP/PCL was faster thanpure PCL which may be due to nHAP/PCL having higher hydrophilicity and facilitating 27
  28. 28. water infiltration. Both types of materials displayed relatively stable mechanicalproperties and stable pore interconnections. This composite showed slight mechanicalweakness to pure PCL but both scaffolds could provide sufficient compressive strengthto handle load-bearing situations. Overall, the investigation shows that in vitrodegradation of the composite remains promising after a six month submersion. A similar study involving nHAP, biphasic calcium phosphate (BCP) and PCL wasreported 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 studyfocused on the nHAP shape and size along with the scaffold mechanical properties,biological degradation, and osteogenic potential. The in vitro bioactivity of the materialwas 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, threedifferent types of nanoparticles were used (needle, rod and spherical shape). Figure 5of this report shows the various types of nanoparticles used in the study. Needleshaped nHAP have an average dimension of 25 nm by 110 nm; the spherical nHAP hasa narrower particle size of 30 nm and rod shaped particles have an average width of 17nm and length of 41 nm. Results indicate that BCP with needle nHAP had acompressive strength of 2.1 MPa, rod-like nHAP particle samples had a compressivestrength of .9 MPa, and spherical shape nHAP particle samples have a compressivestrength of 1.4 MPa. No coating BCP had a compressive strength of .1 MPa, micronHA/ PCL coated BCP and .55 MPa for the PCL coated BCP sample, whereas pure HAonly had a .1 MPa strength. The osteogenesis of the nHAP needles also displayed thebest induction properties. 28
  29. 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/nHAPspherical 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 themelt-molding and particulate leaching methods. The studies focused on porousscaffolds of pure PLGA, nHAP/PLGA at various nHAP wt% and HA/PLGA at various HAwt%. Regarding mechanical strength results, the findings indicate that 20 wt % nHAP /PLGA had the greatest compressive strength at 2.31 MPa. The compressive strengthweakened when the nHA wt % was 40. The 10 wt % nHAP/PLGA showed highercompressive strength than HA/PLGA but the difference was slight. The porosity of the40 wt% nHAP/PLGA was 86; pure PLGA had a porosity of 87% and HA/PLGA had aporosity of 88%. Surprisingly, the 10 wt% and 20 wt% nHAP/PLGA had porosity at 91and 93%, respectively. These two samples also showed better cell proliferation afterthe samples underwent seven days in SBF compared to the 40 wt% nHAP/PLGA. AllnHAP/PLGA samples show better cell proliferation than the HA/PLGA sample and purePLGA sample. This may be due to the homogenous spread of nHAP over HA particlesor the slower degradation rate of the nHAP materials. The absorbancy of the 10 and 20wt% nHAP/PLGA materials also relates to the improved osteoconductivity. The study isfurther discussed in the In Vivo section below. 29
  30. 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 thatresearch will continue to escalate with nHAP as scientists become more articulated withthe 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 studiedbecause it is known to have better performance results than hydroxyapatite (HA). BAGhas been used as bone filler material, applied in clinical treatment of periodontaldisease, and used to replace damaged middle ear bone. This filler can serve as areinforcing component to enhance the stiffness of polymer composites. Compared withmicron-sized bioactive ceramic particles, nano-sized particles have a large specificsurface 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 endowpolymer scaffolds with biomineralization capability but also increase the stiffness ofpolymer material without greatly decreasing the mechanical strength [25]. 30
  31. 31. A few reported studies using nano-bioactive glass ceramic particles (nBGC) wereconducted by M. Peter et al. Both of these studies focused on the inclusion of nBGCand chitosan, a biopolymer derived from partial deacetylation of chitosan. [10, 26]. It isreported that an alternative to tissue regenerative engineering is cell based tissueengineering. This theory is primarily based on the studies that have proven thatnanophase ceramics, compared to microphase ceramics, have better cell-materialinteractions. Chitosan is known to support cell attachment and proliferation because ofits chemical properties. The aim of both studies was to form a composite with chitosanand improve the mechanical and biological properties of the chitosan through theincorporation of bioceramic nanoparticles. The analysis in one of their studies focused on chitosan gelatin (CG) and nBGC withanalysis focusing on the morphology, porosity, mechanical strength and degregation ofthe material [10]. The results show the pore size of the nano-composite scaffolds variedfrom150 to 300 µm, which is adequate for cell migration into the interior regions of thescaffolds. There was also a decrease in the pore size when the chitosan concentrationincreased. The degradation rate was significantly decreased with the addition nBGCwhich may be due to neutralization of the acidic degradation products of chitosan by thealkali 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 bioactivestudies 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 promisingmaterial for bone tissue engineering. 31
  32. 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 materialthat 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]. Theprepared composite particle size was reported at 100nm. The study focused on theswelling, density, degradation and in vitro biomineralization of the material. The swellingis important in a pore because it can aid in the supply of nutrients and oxygen to theinterior regions of the scaffold. It was found that the swelling of the scaffold can becontrolled by the amount of nBGC material in the composite. The biodegradation onnBGC / chitosan, compared to chitosan, is much steadier and slower over time.Furthermore, in vitro biomineralization studies show the deposition of minerals on thesurface 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 compositematerials for bone regeneration. In a study by Z. Hong et al., BGC nanoparticles prepared via a three-step sol-gelmethod with PLLA were found to exhibit bioactive properties [25]. The analysis of thestudy included the porosity of the materials, weighed at different time intervals afterbeing soaked in SBF, as well as SEM, XRD, and TEM analysis of the materials. 32
  33. 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 invitro analysis is plasma body solution. The degredation of the material was monitoredby water uptake, weight loss and pH variation in the medium. It was observed that afterone day of immersion, compared with PLLA porous scaffold, the water content of all the 33
  34. 34. PLLA/BGC composites increased with the introduction of BGC nanoparticles, whichhave a hydrophilic character. A mass increased of 600% is observed for pure PLLAfoam, while for PLLA/BGC composites, this increased ranged from 650 – 800%. PLLAwith 10%wt BGC composite exhibits the highest water absorption throughout the wholeincubation period. The water uptake ability of the nano-composite gradually decreaseswith further increase of filler loading due to the decrease in porosity at higher filercontents. Overall, the researchers found that the inclusion of BGC nanoparticles couldincrease 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 etal. in regards to the development, characterization and in vitro bioactivity of the material[27]. Bioactive glass nanoparticles were prepared by a quick alkali-mediation methodfollowed by a sol-gel process to create the bioactive glass/PLA material. The particleswere controlled to the range of 20-40 nm through adjustment of the pH level in the solammonia solution. The characterization of the study included the porosity, degradation,and in vitro bioactivity of the material. The results show the density of the PLAincreased with the introduction of sol-gel bioactive glass filler and the porosity of thecomposite samples decreased with the increase of the glass content. The weight loss 34
  35. 35. of the material was shown to increase with both the time soaking in SBF and with theincrease of the glass content. Results also show that a negligible amount of calcium and phosphorous was foundon the PLA after soaking the material in SBF for thirty days. On the other hand, thenBGC/PLA materials showed there was a layer of spherical particles on the surface butthe material was still calcium-deficient. Such deficiency could be due to the poor abilityof HA to attract silica ions because of the glass particles. Overall, the results show thatthe addition of sol-gel bioactive glass nanoparticles enhanced the bioactivity of thescaffold and improved the ability to form an HA apatite layer on the surface. A follow-upresearch project is determining how to improve the mineralization of the material byadjusting the nBGC content.V.III. Other composite materials The use of PCL has also received attention as a composite material when pairedwith 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 spiralinterior and a rigid outer tubular part. [28]. The outer part was fabricated with PLGAsintered microparticles and the inner part consists of nanfibre-coated highly porous thinPCL sheets. This design allowed for cells to grow completely across the thin scaffoldwalls and allow for nuturient supply and waste removal. Nanfibres were also depositedon the surface of the spiral structured scaffold to serve as extra-cellular matrix (ECM)mimics for cell proliferation. The incorporation of nanfibres also promotes tissueregeneration through cell attachment, proliferation and differentiation. There were four experiment groups reported: PLGA cylinder scaffold; PLGA tubularscaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubularscaffold with PCL nanfibre containing spiral structured inner core. The results showsome variation between the different designs but, for the most part, show very similar 35
  36. 36. characteristics regarding porosity and mechanical strength which put the design andmaterial 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) compressivestrength mechanical property of PLGA sintered tubular scaffolds with and without insert (ID = 2 mm) [28]. 36
  37. 37. Composite materials have been heavy reviewed recently for bone tissueregeneration because of the ability to balance the strengths of the paired polymermaterial, which possess strong porous behavior and biodegradability to ceramics whichtypically have better mechanical strength and better biocompatibility. Nano-materialsare continually being researched and are in infancy stages with regard to bone tissueengineering. Multiple research projects have incorporated in vitro analysis of materialsbut few projects have included in vivo analysis, which is likely due to the uncertainty ofthe 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 materialswill 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 willserve 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 fluidor plasma solution with a review of the porosity, water uptake of the material afterdesignated periods of time (typically 1, 7, 14, and 28 days), ion retention / mineralizationand degradation of the material to determine if the material is hydrophilic and whether ornot it would serve as a strong candidate bone tissue regeneration material. A few in vivo studies have been conducted with micro based materials but fewstudies have been published using nano-materials, mainly because this research is verycurrent. One of the early reported studies involved nHAP/PLGA, conducted by Y. Cui etal., 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 mostosteoconductive, 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 andfilled 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 bonedefects with the experimental materials experienced various results. Bone callus 37
  38. 38. emerged at 4 weeks post-surgery in the HA/PLGA, 10 and 20 wt% nHAP/PLGA and byweek 24, all samples containing HA or nHAP were bridged by new bones. The 10 and20 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-shapedmononuclear (osteoblast) cells around the bone ossein. The pure PLGA showed theweakest results for specimens that were filled with a bioactive material. The overallconsensus of the study indicates that nHAP/PLGA serves as a promising material forbone 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, Hand L) days.[24]. 38
  39. 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 bonetissue regeneration was carried out and reported by Y. Liu et al. [29]. In their study, theirgoal was to develop a composite scaffold that had a porous structure and similarcomposition to natural bone. The team compared two different types of materials. Thefirst material was a gelatin / nHAP created by glutaraldehyde chemical cross-linking agelatin aqueous solution with nHAP granules at a 5:1 ratio. A second material using thesame gelatin / nHAP base but includes a fibrin glue (FG) mixed with recombinanthuman bone morphogenetic proteins (rhBMP-2) infused into the gelatin/nHAP scaffoldand lyophilized was used. The test subjects of the study were fourty-five adolescent New Zealand whiterabbits. After the rabbits were anesthetized, a segmental defect was made on themiddle radial diaphysis of the foot. The defect was irrigated and filled with either the 39
  40. 40. first material or second material mentioned above or nothing. The animals underwentX-ray examinations at 4, 8, and 12 weeks. The findings of the study showed that nHAP particles were homogeneously localizedin the gelatin walls of the gelatin/nHAP scaffold and the porosity was 91.4% withcompression strength of 1.32 MPa. The pore diameter of the gelatin/nHAP/FG scaffoldwas 87.9% with a compressive strength of 1.98 MPa. A significant result shown is thatthe gelatin/nHAP/FG scaffold has better bioactivity over a longer period of time andreleases at a steady rate over the course of the 40 days compared to the gelatin/nHAPmaterial that was completely released within half this time. This slower release allowedfor 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 scaffoldmaterial 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 appliedin bone tissue regeneration. This study, along with others, demonstrates a wave ofopportunity in both the sciences and in the well-being of advancing the generalpopulation. Studies should be conducted to determine how the various materialsperform when not only bone needs regeneration but also when cartilage andsurrounding ligaments and perhaps muscle need to be regenerated. Strides in thisdirection will become extremely valuable to intellectual property as well as advancingmedical sciences and the general welfare of man. 40
  41. 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 bestmaterial(s) to use in bone tissue regeneration. There is a great opportunity to servesociety with solving issues related to unexpected fractures of the young, predictablefailings of old tissues, such as cancer and osteoporosis, found in the elderly andreplacing the current process of prosthetics implantation failure through this research. Cancellous bone tissue is composed of 70% hydroxapatite nanoparticles andapproximately 30% type I collagen. Although tissue can naturally repair itself whennano, and occasional micro, damage is done, bone tissue cannot heal itself naturallywhen milli or larger damage is inflicted. Therefore, advanced research in implantationregenerative materials are sought. The qualities desired for an optimal scaffold materialare: bioactivity (ability to bond to bone), osteogenic (stimulation of bone growth),biocompatible (induce minimal toxic or immunie respone in vivo), resorb safely andeffectively 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. 42. requirements for clinical use. Research has been conducted on polymers, ceramicsand composites. A number of different fabrication methods have also been created andoptimized for the different materials. The focus of this study was to: gain a general understanding of how bone tissue isnaturally formed and how nano-technology plays an important role in the developmentof finding a suitable bone tissue regenerative material, understand the main materialsutilized in bone tissue regeneration, and comparing the mechanical strength to porosityof materials that utilize nano-particles. The research conducted in this field is verycurrent with most of the findings by different research teams reported within the past sixto twelve months. The overall development of this type of research is proving itself tobe very beneficial with a great deal of opportunity still available to learn about the in vivoperformance 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 includepoly(lactic acid) (PLA) and poly(glycolic acid) (PGA), and poly(lactic-co-glycolide)(PLGA) for synthetic polymers [12]. Polymers can be very porous and biodegradablebut are problematic due to potential toxicity with degrading too quickly and poor materialstrength. Ceramics make a better cancellous replacement material to promote boneregeneration. HA is a natural solution and material choice because of the bone’scomposition. HA nano-particles attract the cells to attach and form of extracellularmatrix within the porous tissue. However, HA does not work well individually because itis brittle and fractures easily. Bioactive glass is also a ongoing researched materialbecause of its bioactivity and osteoconductivity. 45S5 Bioglass®, which is composedon 45% , 24.5% , 24.5% and 6% in weight percent, is one of thepredominant bioactive glass compositions utilized. Although neither a polymer nor a ceramic works well individually as a bone tissuematerial for regenerative purposes, the composite of the different materials are being 42
  43. 43. proven as the best solution to fit all of the necessary attributes. The focus of this paperwas to compare and evaluate the porosity to the compressive strength of differentmaterials that incorporate nano-particles. Table 4 provides a summary of the porosityand compressive strength of different materials currently under investigation. Thesefindings are also composed in Graph 9. CompressiveMaterial Porosity (wt %) Strength (Mpa) ReferenceCancellous Bone 70 4 7Cancellous Bone 90 0.2 7Porous Diopside 75 1.36 8Porous Diopside 90 0.2 845S5 Bioglass / nHAP 60 1 17Shell Scaffolds 80 - 1813-93 Bioglass 85 11 15nHAP / PLA 86.2 0.44 16nHAP / PLA 91.5 0.29 16PLLA 87.5 1.8 21nHAP / PLLA 85 8.5 21HA / PLLA 86.5 4.6 21nHAP needle / BCP / PCL 91 2.1 23nHAP rod / BCP / PCL 91 0.9 23nHAP sphere / BCP / PCL 91 1.4 23BCP 94 0.1 23HA / BCP /PCL - 0.55 23nHAP 20% / PLGA 93 2.31 24nHAP 10% / PLGA 91 1 24nHAP 40% / PLGA 86 0.75 24HA / PLGA 88 0.8 24PLGA 87 1.1 24BGC 10% / PLLA 92 0.34 25BGC 20% / PLLA 91 0.33 25BGC 30% / PLLA 88 0.35 25PLLA 92 0.28 25PLGA cylinder / PCL 30 9.22 28PLGA tubular / PCL 35 9.8 28PLGA tubular / PCL spiral 46 9.9 28PLGA tubular / PCL nanofibre 44 9.1 28 Table 4: Summarized of various composites using nano particles and compares to micro particles. 43
  44. 44. 12 Porosity v. Compressive Strength Cancellous Bone Cancellous Bone Porous Diopside Porous Diopside 10 45S5 Bioglass / nHAP 13-93 Bioglass nHAP / PLA nHAP / PLACompressive 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. 45. VIII. Bibliography[1] L. Zhang, “Nanotechnology and nano-materials: Promises for improved tissueregeneration,” Nano Today, Vol. 4, pp. 66 – 80, (2009).[2] J. Venugopal et al., “Biomimetic hydroxyapatitie-containing composite nanofibroussubstrates 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 CeramicSociety, Vol. 29, pp. 1257 – 1265, (2009).[4] M. Wang, “Composite Scaffolds for Bone Tissue Engineering”, American Journal ofBiochemistry 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 bonetissue engineering”, Acta Biomaterialia. (2010), doi: 10.1016/j.actbio.2009.12.022.[9] J. Jang et al., “Electrospun materials as potential platforms for bone tissueengineering”, Advanced Drug Delivery Reviews, Vol. 61, pp. 1065 – 1083, (2009).[10] M. Peter et al., “Novel biodegradable chitosan–gelatin/nano-bioactive glass ceramiccomposite scaffolds for alveolar bone tissue engineering”, Chemical EngineeringJournal, Vol. 158, pp. 353 – 361, (2010). 45
  46. 46. [11] M. Swetha, et al., “Biocomposites containing natural polymers and hydroxyapatitefor 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/inorganiccomposite scaffolds for bone tissue engineering,” Biomaterials, Vol. 27, pp. 3413 –3431, (2006).[13] V.S.W. Chan, “Nanomedicine: An unresolved regulatory issue”, RegulatoryToxicology 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 scaffoldsprepared 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-sizefiller 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 tissueengineering: 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 strengthbioceramic scaffolds for bone regeneration”, Materials Letters, Vol. 64, pp. 203-206(2010).[19] O. Suzuki, “Octacalcium phosphate: Osteoconductivity and crystal chemistry”, ActaBiomaterialia, (2010), doi: 10.1016/j.actbio.2010.04.002. 46
  47. 47. [20] X. Cai et al., “Preparation and characterization of homogeneous chitosan-polylacticacid/hydroxyapatite nano-composite for bone tissue engineering and evaluation of itsmechanical properties”, Acta Biomaterialia, Vol. 5, pp. 2693 – 2703 (2009).[21] E. Nejati et al., “Needle-like nano hydroxyapatite/poly(L-lactide acid) compositescaffold 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”, PolymerDegradation and Stability, Vol. 95, pp. 207 – 213, (2010).[23] S. Roohani-Esfahani, “The influence hydroxyapatite nanoparticle shape and size onthe 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) andhydroxyapatite surface-grafted with L-lactic acid oligomer for bone repair”,ActaBiomaterialia, 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 nanoparticlesdisseminated chitosan matrix for tissue engineering applications”, CarbohydratePolymers, Vol. 79, pp. 284 – 289, (2010).[27] A.M. El-Kady et al., “Development, characterization, and in vitro bioactivity studiesof sol-gel bioactive glass/poly(L-lactide) nano-composite scaffolds”, Material Science andEngineering C, Vol. 30, pp. 120 – 131 (2010). 47
  48. 48. [28] J. Wang & X. Yu. “Preparation, characterization and in vitro analysis of novelstructured 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-loadedgelatin/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 bonetissue engineering”, Advanced Drug Delvery Reviews, Vol. 61, pp. 1065 – 1083, (2009). IX. AppendixIX.I Fabrication methods available There is a wide array of fabrication methods available and used for the differentmaterials available for bone tissue engineering. The choice of a fabrication methoddirectly impacts the material’s physical properties, including its porosity and mechanicalstrength. The available fabrication methods all have strengths and weaknesses to itsuse. Such strengths and weaknesses are outlined in the following table.Fabrication Mode Advantages DisadvantagesThermally induced phase High porosity (~95%), Long time to sublime solvent (48separation 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 solventsSolvent 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. 49. Protein and cell encapsulation possible, Some methods use organic Good interface with medical imaging solventsMicrosphere sintering Graded porosity structures possible, Interconnectivity is an issue, Controlled porosity, Use of organic solvents Can be fabricated into complex shapesSol-gel / Foam Sol-gel Easy to control Long time to create / multiple steps to formElectrospinning 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 fabricationtechniques impact different materials based on the molecular composition of thematerial utilized. Although it was outside the scope of this paper to analyze the differentforms of fabrication, a brief outline of the preferred (or utilized) method of fabrication fordifferent 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. 50. IX.II Nano-fibresNano-fibres is a broad type of nano-material produced in a particular fashion by itsfabrication process. Nanfibres are typically created through electrospinning. There is agreat deal of research already conducted on this topic. This form of material formationwas lightly reviewed in this report because of the current trend of novel researchfocused on nanoparticles rather than nano-fibres. Nevertheless, a table is attachedwhich shows the wide variety of materials that can be spun into nanfibres for use asbioactive scaffolds. [30]. Figure 14: Summary of Electrospun nanfibre materials for bone reconstruction [30]. 50

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