Biomaterials for medical

applications
Implants, tissue engineering
Drug delivery systems

Dr. Gisela Buschle-Diller

Auburn University
 Biomedical materials

Implants: stability in Biointegratable materials:

biological tissue engineering, sutures;
environments degradation in biological
environments

Drug delivery devices

Biomedical applications
GENERAL ASPECTS, IMPLANT
MATERIALS, TISSUE ENGINEERING
 Aspects to consider

I. Armentano et al., Polym. Degrad. Stabil. 95, 2010, 2126-2146

 Materials commonly used for medical
applications

Silicone
PET
PMMA
Gold Very different Different proteins adsorb
Polyurethane materials at their surfaces
Stainless steel (more than 200 proteins)
Rubber
Biopolymers
….
Foreign body response:
Message: “foreign material (trash) must go out”
 Bioresponses
• State of a cell depends on signals it gets from the
environment: for example, platelets circulate passively
in blood stream – upon an injury, signals are received
that activate their role in healing process

Very difficult to study on isolated cells and

biomaterials, because the biological system functions
as a whole; model systems usually do not suffice!

Biomaterials are needed that direct biological healing

responses (that interact with the cells)
 Biocompatibility
• First body response is encapsulating any biomaterial
in a collagenous bag (“foreign body response”) to rid
itself of the biomaterial and to avoid contact of
device and tissue

Read Tutorial – very good!!!

http://uweb.engr.washington.edu/research/tutorials
/woundhealing.html
U Washington Engineered Biomaterials
 Cells (neutrophils and macrophages)
Adsorbed proteins (1 min) interrogate the biomaterial (1 day)

Implant

Collagen

Cells form giant cells and send protein

signaling agents (cytokines)

In response fibroblasts arrive

and make collagen

Biomaterial enclosed in collagen bag (1-3 weeks)

 Questions
• Why does the body not heal normally around
implants? – Still not clear
• How to work around foreign body response (hide the
implant…)?
• How to study this response outside the body (in
vitro) as opposed to inside (in vivo)?
 To avoid foreign body response
• Strategy #1: Surfaces modified for better
recognition (acceptance)

• Strategy #2: Surface modification to avoid

non-specific adsorption of proteins
 Initial body response to injury/implant

Replacement material
Within seconds
Most important
Protein layer formed

Cell adsorption

Primary plasma proteins: albumin (60-70% of plasma),

immunoglobulins (20%); fibrinogen; albumin seems to coat the
implant; fibrinogen denatures, then causing increasing adherence of
immunoglobulins
 Possibility 1: modify biopolymer
surface

• Greater texture and porosity give better surface

interaction
• Chemical composition of surface can ease
adsorption of desired proteins
– Surface could be modified after polymer is formed
– Surface functional groups could be introduced
through use of functionalized monomer
• Hydrophobic surfaces bind more (hydrophobic)
protein
• Surface potential influences ion distribution and
interaction with protein
 Possibility 2: target specific proteins
for adsorption

• Larger proteins have more surface contact

• Less crosslinked (less stable) proteins unfold
easier and make more contact possible
• Close to isoelectric point (same number of
positive and negative charges) proteins adsorb
more readily
Reality check in-vitro versus in-vivo
• Blood interaction: implant testing requires a
wound – blood is the first contact with
implant
• Blood (plasma, various kinds of cells, platelets)
• Implant surface properties determine which
proteins will be adsorbed first and at what
concentration
• Protein can undergo conformational change
upon adsorption or can be desorbed
 From the biomaterial side:
• Synthetic biopolymers for tissue engineering
or implants lack biological cues for cells to
adhere or proliferate
• Nanocomposite materials from natural
polymers with synthetic biopolymers could
help improve the situation
• Coating of natural on synthetic biopolymers
 Common practice of tissue engineering

Cells cultured on natural tissue, then tissue implanted into patient

Tian et al., Progress in Polymer Sci. 37, 2012, 237-280.
Biomaterial options
NATURAL PROTEINS FOR TISSUE
REPAIR
 Natural choice: collagen
• Chief structural scaffolding of vertebrate body
(25-30% of total body proteins): skin, bone,
tendon, connective tissue, cartilage, membranes,
cornea…
• Family of distinct compounds (16-19 types) made
in unique triple-helix configuration of 3
polypeptide subunits known as α-chains;
different in length of helix and size of non-helical
portion of the molecule
• Responsible for strength
 Collagen structure

http://www.ncbi.nlm.nih.gov/books/NBK21582/
Very good summary of the most important issues related to collagen
 Primary structure: amino acids (α chains);
polypeptides

Secondary structure: left-handed helix

Tertiary structure: triple helix

Quaternary structure: staggered triple

helices

Normal collagen After arthritis

Friess, W., Europ. J. Pharma. Biopharma., 45 (1998), 113-136.

 Collagen properties and applications

• Biocompatible, biodegradable, nontoxic; good

interaction with other biopolymers and natural
materials (e.g., with other proteins); forms films,
sheets, beads, sponges
• Difficult to dissolve
• Difficult to obtain in reliable way
• Biomedical uses: drug delivery, sponges for burn
wounds, tissue engineering, artificial skin, tablets,
etc. (a collagen film applied to the human eye is
decomposed/incorporated within 5-6 h)
Electrospun collagen mat

Collagen type III (calf skin)

dissolved in 1,1,1,3,3,3-hexafluoro-2-
propanol (HFP) and electrospun

Tested with bovine cells

Problem: traces of solvent and

sterilization

Student senior design

project 2004
 Collagen blends with other FDA approved
polymers for medical applications

• Collagen blends with PVA, PVP, PEG and others

(if common solvent can be found):
– Wound dressing
– Tissue engineering; biomedical scaffolds, artificial
skin
– Cell adhesion improved if collagen is present
• Collagen blended with polyurethane also
possible
 Drug release from collagen
• Thin films or sheets: drugs held by hydrogen bonding
or entrapment
• Combinations of collagen and elastin for drug release
• Collagen sponges by combination with elastin and
fibronectin; crosslinked or not; wound management
• Formation of small drug-loaded collagen discs for
release of antimicrobials to the cornea

www.bmglabtech.com
 Guided tissue regeneration (GTR)
membranes
• Idea: create membrane based on collagen that guides
tissue regeneration by exclusion of unwanted fast
growing cells
• Currently: Teflon® membranes (PTFE,
polytetrafluoroethylene) – good, but non-biodegradable;
require additional surgery to remove
• Replacement of Teflon with collagen membrane: several
commercially available
• Collagen membranes biodegrade too fast
• Crosslinking of membranes leads to inert material that
prevents biointegration

Abu Neel et al., Adv. Drug Delivery Rev. 65, 2013, 429-456
 Example of commercial collagen
membrane
Bio-Gide®, Geistlich, Switzerland: double-layer of collagen
to delay biointegration

Guided tissue and bone regeneration (dental)

www.geistlichonline.com
 Fibronectin, fibrinogen and fibrin

• Fibronectin: non-collagenous adhesive protein;

enabling cells to attach to the extracellular matrix;
anchoring of cells to collagen -> important protein for
tissue engineering and repair; rod-like structure with 3
types of sections
• Fibrinogen: plasma protein produced in the liver;
important in blood clotting to stop bleeding
• Fibrin: also plasma protein important during blood
clotting – formation of a fibrillar network at the site of
a wound (too much fibrin can lead to thrombosis)
 Collagen-fibronectin
• Fibronectin (1% of serum proteins) assists
communication with cells; important for
binding of a cell to collagen
• Application example: three-dimensional spinal
cord constructs from collagen-fibronectin:
– Collagen formed into a hyper-gel and co-
compressed with fibronectin to remove liquid
– collagen forms outer tube, fibronectin the inner
core
 Fibrinogen to fibrin
• Protein produced in the liver (3 paired
polypeptide chains)
• Fibrinogen helps to stop bleeding by formation of
blood clots
• Primary role: in the final stage of coagulation
fibrinogen is converted to fibrin (clot)
• Fibrin forms a firm insoluble gel by crosslinking of
the protein polymer and limits bleeding
• Dysfunction: excessive bleeding or thrombosis
 Blood coagulation

http://tollefsen.wustl.edu/coagulation/coagulation.html
 Fibrin as natural glue

http://www.refractiveeyecare.com/2012/03/o
phthalmic-applications-of-fibrin-glue/
 Elastin
• 70% (dry) of elastic material (arteries, lungs, skin,
cartilage, ligaments, etc.) for elasticity and resilience
• Limited understanding (and practical application) due
to difficult extraction of elastin from native tissue

CH2-NH-CH2 crosslinks
Triple helix

stretch

relax

Covalently crosslinked network Alignment

(coiled unorganized peptide chains)
 Silk fibroin sponge

Animal protein fibroin and sericin

SILK IN BIOMEDICAL APPLICATIONS
 Silk mechanical properties
Material Modulus (GPa) Elongation (%)

Silk (B.mori), no 5-12 19-20

sericin
Silk (B.mori), with 15-17 4-6
sericin

Orb spider silk 11-13 17-18

Collagen 0.0018-0.046 24-68

Crosslinked collagen 0.4-0.8 12-16

PLA 1.2-3 2-6

 Fibroin properties
• Good biocompatibility
• High mechanical strength: strength to density
ratio up to 10 times that of steel!
• Very slow biodegradation
• Regenerated fibroin from salt or ionic liquid
solutions:
– biodegradation fast, but adverse reactions in body
might occur
– Films with drugs loaded
– Hydrogels
– Porous scaffolds by freeze-drying
 Biomaterials made from fibroin

Hydrogels,
Powder
Tissue scaffolds
Fibers
Particles

B. Kundu et al., Adv. Drug Delivery Rev. 65, 2013, 457-470.

 Medical uses of silk fibroin
• Non-absorbable suture threads with/without
modification, with/without wax or silicon coating
• Wound dressing (nonwoven mats)
• Tissue engineering scaffolds based on films from
regenerated silk solution, seeded with cells or
reinforced with ground silk
• Cornea tissue repair materials
• Stents for artery replacement (but strength not
very good yet; high cost)
• Nerve grafts, but limited availability; deformation
issues
Lawrence et al., Biomaterials 30, 2009, 1299; Kim et al., Nature Mater. 9, 2010, 511.
You invented a new biopolymer and
want to sell it for suture threads.

What would be the most important

properties it should have?

Please interview your neighbor

(2 min each)
Poly(hydroxy alkanoates) (PHA)

BIOPOLYESTER IN MEDICAL
APPLICATIONS
 Most important aliphatic biopolyesters
Name Short name Source
Poly(glycolic acid) PGA Petrochemical (biomass,
(2 C) microbial not economic)

Poly(lactide) PLA Biomass, corn

(3 C, branched)

Poly(hydroxypropionate) PHP Mircobial from sugar (in

(3 C, linear) cell as storage)

Poly(hydroxybutyrate) PHB Microbial from sugar (in

PHAs (4 C) cell as storage)

Poly(hydroxyvalerate) PHV Microbial from sugar (in

…. More PHAs (5 C) cell as storage)

Poly(ε-caprolactone) PCL Petrochemical (biomass,

…. (6 C) microbial not economic)
 Conditions for tissue engineering
• Biocompatibility
• Support of cell growth and adhesion
• Guide and organize cells
• Allow in-growth of cells, passage of nutrients
and waste products
• Biodegradable without forming toxic products
• Desired biodegradation time frame
 Medical applications of PHAs
• Wound management (swabs, skin replacement,
sutures, staples
• Orthopedic (scaffolds, bone grafts, meniscus
regeneration, screws)
• Dentistry (tissue regeneration in periodonitis)
• Vascular systems (heart valves, patches, vascular
grafts)
• Drug delivery (spheres for anticancer therapy,
antibiotics)
 PHA-copolymer films

Films used to grow cells for tissue engineering

Chee et al., Biochem. Eng. J. (2008) 38(3), 314
 PHA-films loaded with drugs

No drug 10% drug loading 40% drug loading

Chee et al., Biochem. Eng. J. (2008) 38(3), 314

PHA-films with cell growth
 Implants materials made from PHAs

Chen, Wu, Biomaterials (2005) 26(33), 6565

Polylactide
PLA AS BIOMEDICAL MATERIAL
 PLA as biomedical material
• FDA approved biopolymer; biodegradable by enzymes or
hydrolytic conditions; natural metabolites in the body
• Degradation rate tailored by crystallinity (PLLA or PDLA) –
important for drug delivery
• However: during biointegration the pH changes due to
formation of an acid
• Good mechanical properties
• Applications in regenerative medicine, suture threads,
drug delivery
• Amphiphilic blocks with other biopolymers (natural and
synthetic) for increased hydrophilicity where needed
• PLA-copolymers with PGA for targeted cancer therapy
 Market share of PLA medical devices fairly
low – why?

To anchor implants in bone:

PLA screw (top), 70% PLLA,
30% PDLA, Resomer®
(Boehringer); titanium and
PLA (bottom)
PLA market distribution 2012

NIH.gov website www.gii.co.jp

Poly(glycolic acid) PGA, poly(ε-caprolactone) PCL
ALIPHATIC BIOPOLYESTERS OTHER
THAN PLA AND PHA
 Most important aliphatic biopolyesters
Name Short name Source
Poly(glycolic acid) PGA Petrochemical (biomass,
(2 C) microbial not economic)

Poly(lactide) PLA Biomass, corn

(3 C, branched)

Poly(hydroxypropionate) PHP Mircobial from sugar (in

(3 C, linear) cell as storage)

Poly(hydroxybutyrate) PHB Microbial from sugar (in

PHAs (4 C) cell as storage)

Poly(hydroxyvalerate) PHV Microbial from sugar (in

…. More PHAs (5 C) cell as storage)

Poly(ε-caprolactone) PCL Petrochemical (biomass,

(6 C) microbial not economic)
Biodegradable aliphatic polyesters PGA
and PCL made from petrochemicals
O

O Poly(glycolic acid) PGA

PGA

O Poly(ε-caprolactone) PCL
n
O
PCL
 PCL and PGA
• Biopolyesters – biodegradable, compostable,
bio-integration into the body possible (FDA
approved)
• Not from renewable resources (amounts from
microbial production is too small)
• Very important for biomedical applications
 Essential characteristics for biodegradable
polymers for medical applications

• Both PGA and PCL (and their decomposition

products) are biocompatible
• Rate of degradation can be matched to the rate of
new tissue formation during the healing process
• PGA, PCL and degradation products approved by FDA
• Processing with common equipment possible and
solubility in common solvents
• Economic shelf life
• Sufficient strength
 PGA – not available from renewable
resource

O O
O ring-opening polym.
O C C
O H2
O n

PGA
glycolide (di-ester)

Currently obtained from petroleum (catalysts: Sn- or Al-alkoxides)

Small scale production by use of lactobacillus, similar to lactic acid

 Properties of PGA
• High melting point (225°C)
• Glass transition at 35-40°C
• High MW PGA is difficult to dissolve in any solvent;
crystallinity fairly high at 45-55%
• Soluble in fluorinated solvents – formation of fibers
possible – medical applications (sutures)
• More useful in form of co-polymers, for example
with PCL (poly(glycolic-co-ε-caprolactone)) or PLA
(PGLA) – different ratios of PGA and PCL determine
solubility and time for degradation in the body
• Very useful for sutures
 Nonabsorbable suture threads
Material Origin Absorbable Application
Nylon Nylon 6 or 6.6 No (dyed or clear) Soft tissue,
ophthalmic,
neurological

Polyester PET, coated No (dyed green) Soft tissue,

orthopaedic
Polypropylene Isotactic No (dyed or clear) Soft tissue,
polypropylene orthopaedic,
cardiovascular
Stainless steel No Orthopaedic,
abdominal wounds

Silk Fibroin from No (dyed black) Soft tissue, ligation,

Bombyx mori silk; cardovascular, etc.
w/wt sericin
 General sterilization methods

• Steam and pressure (>150°C, 20-30 PSI); 5-30

min, autoclave
• Dry heat at higher temperature and longer
• γ-irradiation
• Ethylene oxide gas exposure (very toxic)
• Soaking in cold disinfectant solution for 10-20
min

See WHO Pharmacopoeia Library for details

 Absorbable suture threads
Material Composition Absorbable Application
Surgical gut Sterile connective Yes (fast/slow Soft tissue
tissue of beef or depending on
sheep (collagen) type)
PGA 100% PGA Yes (60-90 days) Soft tissue
ophthalmic
PGA-PCL PGA-co-PCL Yes (strength loss, Soft tissue, facial
variation on starting after one wounds,
copolymer ratios week; integration pedriatric
(90:10 to 50:50) 91-120 days)

PDS Poly(-p-dioxanone) Yes, 4 months transdermal

poly-ester-ether
 Biodegradation paths

PGA PDS PLA

Glycolic acid Glyoxylate

Glycine Lactic acid

Serine
Excreted in urine
Pyrovate
Acetyl coenzyme A
Caution: acidic intermediate
biodegradation products Citric acid cycle

CO2 and H2O

 Commercial products of PGA suture
threads
• Most common: suture threads (made of 100%
PGA) – absorbable, can be made sterile
– PGA sutures: 84% of initial strength retained after
2 weeks; 51% after 3 weeks; 24% after 4 weeks;
total absorption time 60-90 days
– Smooth coating of thread for easier tissue passage
and minimal tissue irritation;
– Precise knot formation and knot lie-down
PGA surgical threads

Braided or not
Needles bent or straight
 PGA as scaffold material

Cells obtained from patient; cell culture on PGA fiber scaffold; scaffold with new
tissue growth returned to patient

www.biology-online
PGA tubes for peripheral nerve repair

100% knit PGA

Break-down in body within
3 months
Completely resorbed 6-9 months

Kink-free, oxygen permeable to

assist nerve regeration

www.synovislife.com
 PGA-PLA copolymers

Fibers

Sponge-like

www.biology-online
Examples of commercial PGLA
products

PLA-PGA barrier membranes: barrier

function up to 4 months
http://www.unicarebiomedical.com
/cytoflex_resorb.htm
Poly(ε-caprolactone) PCL
 PCL – biodegradable, but not from a
renewable resource
O O

O
petroleum

cyclohexanone ε-caprolactone

Conventional industrial synthesis

Ring-opening
polymerization,
O
n also possible
PCL
O using a lipase
 Poly-ε-caprolactone (PCL)
• Injection and extrusion processing possible; good
adhesion properties, miscible with pigments, fillers, other
polymers
• Problem: catalyst for controlled ring-opening process
(metal alkoxide) often remains in the product and leaches
out slowly; less controlled ROP by less harmful catalyst,
such as alcohol; enzymatic by lipase (little control)
• Tg: -60°C; Tm: +60°C
• Medical application: drug release; slow degradation,
slower than PLA; semicrystalline (50%), highly
hydrophobic
 Commercial products made from PCL
• PCL in copolymer products to influence its
degradation rate and mechanical strength
– Example: PGA-PLA-PCL: terpolymer of glycolide (60%)-
lactide (30%)-caprolactone (10%): tough material with
half-life of 15-20 days
• Most applications of PCL are more related to its
low Tg than its degradation
• Commercial products from Solvay (CAPA), Daicel
(Celgren), Dow Union Carbide (Tone)
• Copolymers with other biodegradable monomers
• Drug loading for timed release possible in
biomedical devices
 Typical properties of commercial PCL
Property CAPA 640 CAPA 650 CAPA 680

MW 37,000 50,000 80,000

Melting 58-60 58-60 60-62

point (°C)
Tensile 140 360 580
strength
(kg/cm2)
Elongation 660 800 900
at break (%)
 PCL decomposition
• PCL degrades quickly in various environments
(fungal sludge, compost, enzymes, soil, body,
etc.) - too quick for some applications
• Control of degradation rate: blending, co-
polymerization; molecular weight; porosity;
pore size, distribution; physical form, etc.
• PCL as films, foams and solid products (easily
shaped)
 Biodegradation of PCL

O
n
PCL O
Attack in the amorphous areas
lipase
or cutinase (increase of crystalline ratio)

H OH Broadening of MW distribution
O

ε-hydroxyhexanoic acid
O
Rapid MW decrease
ω-oxidation
O
H OH
O CO2 + H2O

O
 Porous scaffolds from copolymers
of PCL with other polymers

50:50 PCL:PV(OH)

Poly(vinyl alcohol) is water-soluble

and washed out – pore formation

40:60

30:70

Tay et al., J. Materials Processing Technol. 182 (2007), 117.

 Films made from PCL-PGLA copolymers for
improved cell adhesion by patterning

PCL:PGLA 9:1
Pure PCL

PCL:PGLA 7:3
PCL:PGLA 8:2

Modification of surface morphology and hydrophilicity

Tang et al., (2005), Biomaterials 26, 6618.

 PCL foam products
• Differentiation by foam density, cell size and
distribution
• Conventional blowing agents: N2, CO2; pellets
saturated with blowing agent, then heated past the
Tg under pressure

• Microcellular foams with very small cells and very

high density for tough materials, (non-medical: car
parts, aircraft, sporting goods, etc.; better fatigue
than non-foamed polymers)
 Tissue engineering

Microcellular PCL foam for scaffold materials

Luetzow et al., J. Biomechanics 40 (2007), 580.

 Biomaterials as drug delivery
vehicles

What are most important points to

make a system work?

Please interview your neighbor

(4 min)
Some examples
BIOPOLYMERS FOR DRUG DELIVERY
SYSTEMS
 Possible drug delivery systems
• Ingested (capsules, pills)
• Inhaled (micro-, macroparticles)
• Injected (micellar, emulsion, microparticles)
• Ocular (eye drops)
• Transdermal (patch)
• Implanted (reservoir;
matrix/hydrogel)
Actual medicine plus non-active ingredients:
Perservatives, antioxdants, surfactants, buffers, viscosity modifies,
flavors, emulsifiers, sugar, gelatin, cellulose derivates (MC, CMC)...
 What’s important?
• Release at specific target (general pain relief
versus chemotherapy for cancer)
• Release with best concentration
• Non-adverse reactions of ALL ingredients,
active or non-active

• Sterilization?
• Monitoring in the body?
 Requirements for effective drug
delivery
• Drug delivery systems must be stable, non-
toxic, reproducible and well-characterized
• Decomposition must be well-known
– Polymer by itself
– Release characteristics of the drug
– Differences between in-vivo and in-vitro systems
must be known
• Dynamically changing systems during bio-
deterioration
 Possibilities
• Drug in matrix: polymer breaks down over time,
releasing the drug
• Drug attached to polymer backbone: cleavage by
enzyme
• Encapsulated (in gelatin, PEG or other)
• Polymers as drug carriers: enzymatic cleavage
(ester bond hydrolysis) or hydrolytically (pH,
water, etc., stomach pH 1.5-2.5; intestines pH 6.6-
7.5)
• Polymeric drugs: poly(aspirin) in water: salicylic
acid and sebacic acid
Examples of drug delivery systems
• Most studied polymers for implanted delivery
systems: PLA and copolymers (PLGA)
• Most common drugs: anticancer, hormones,
vaccines, antibiotics, proteins, anti-
inflammatory agents, etc.
• Important: hydrophilic/hydrophobic
characteristics
• Consideration: pH change during drug release
possible!
 General drug release profile
Controlled release
concentration

Burst release

max. release

travel time time

application
to target
 Polymer hydrolysis
• Mechanism: either random (at basic pH) or by
unzipping from the chain end (under acidic
conditions); physiological pH (or simulated
physiological): pH 7.4; 37 degrees C
• Chemical reactions induced by mechanical
stress (important for all orthopedic implants)
• Irradiation (gamma rays) and ultrasound:
accelerated breakdown
 Drug release – a dynamic system

• Drug solublitity in
polymer
• Drug solubility in
release medium
(pH, composition)
• Solubility might change over time with
polymer biodegradation products present
• Release mechanism might change with time
Picture: Sigma-Aldrich
PLA electrospun coated with alginate hydrogel
M. R. Abidian, D. C. Martin, Adv. Funct. Mater. 2009, 19, 573. Penn State U.
 Polymer matrix: factors of influence
• Polymer chain length (shorter chains degrade
faster)
• Crystallinity
• Chemical composition
• Production parameters (solvent used; electrolytes
present, stirring, shear rate, type of solvent
removal, etc.)
– Size and porosity of microspheres, surface
morphology, swelling, accessibility of linkages for
degradation
 System parameters
• Polymer, solvent, drug interaction during
preparation of drug delivery device
• Solubility of drug in release medium
• Dynamic system
• Degradation products, lower amount of drug,
impact on entire system
Packaging a drug – some examples

Incorporation of drugs in PEGylated zein micelle

Podaralla et al., Mol. Pharmaceutics, 2012, 9, 2778

 Enzyme responsive nanoparticle
systems

Copolymer breaking apart,

releasing drug

www.discover.positron.edu.au

Rica et al., Adv. Drug Delivery Rev. 64, 2012, 967-978

 What will the future hold – nanosized DNA
boxes to open with key and release drugs?

www.nextbigfuture.com
 Muchas gracias a

Bernal Sibaja Hernández y Vladimir

Quinones Silva (Auburn University)
y
Mariana Miñano

para ayudarme con el español