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Primum non nocere
Primum non nocere
Věda vychází ze systematického studia založeného na
pozorování, tvorbě teorií a jejich ověřování
experimentem.
Ignác Filip Semmelweis:
Horečka omladnic. 1847 zavedl
na oddělení povinné mytí
rukou chlorovým vápnem
William Morton: První
operace v celkové
narkoze - 1846
PIP scandal
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Murphy’s Law
“Může-li se něco pokazit, pokazí se to.”
“Jestli někdo může udělat něco špatně, udělá to tak”.
Nekompatibilita konektorů vedla k smrti dítěte – dodán špatný kabel.
Wieringa. AVOIDING PITFALLS IN THE ROAD FROM IDEA TO CERTIFIED PRODUCT (AND THE HARSH CLINICAL ENVIRONMENTTHEREAFTER) WHEN INNOVATING MEDICAL
DEVICES. Belgian Day on Biomedical Engineering
PIP (Poly Implant Prothèse ) scandal
What is a breast implant?
A medical prosthesis used to augment, reconstruct, or create the physical form
of breasts
Modern (so-called fifth generation) breast implants usually contain either saline
or a viscous silicone gel
The outer casing, or shell, is composed of durable elastic silicone manufactured
through a chemical process called vulcanization in which sulphur is added to
the silicone to increase durability
Due to the production method, commercial silicone products will contain
variable concentrations of molecular weights and sizes including a
subgroup of small-sized molecules referred to as D4, D5, D6. In addition,
the normal manufacturing process may result in traces of platinum, used
as an essential catalyst.
The PIP implants were found to contain a higher proportion of small-sized
molecules D4, D5, D6 than the norm.
https://doi.org/10.1177/0141076813480994
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PIP (Company Poly Implant Prosthese) scandal
Timeline:
1991: PIP launched by Jean-Claude Mas
1997: PIP authorized to produce medical-grade silicone implants
2000: FDA warns about deviations from “good manufacturing practices” found at the
PIP plant. Additionally, the company withdraws its hydrogel implants from the market
when it cannot show they are safe.
2001: PIP starts using unapproved, (“in-house” formula) industrial-grade silicone in
their implants
2009: Concerns surfaced in France when surgeons started reporting abnormally high
rupture rates
2010: PIP was placed into liquidation after the French medical safety agency recalled
its implants.
2011: The French government recommended that 30,000 women with PIP implants
seek removal of the implants as a precaution
2013: Mas sent to prison for four years, fined 75,000 euros, and banned for life from
working in medical services or running a company.
FDA - 2016 Medical Device Recalls
Greatbatch Medical Recalls Standard Offset Cup Impactor Used for Hip Joint Replacement due to
Inadequate Sterilization
Centurion Recalls Multi-Med Single Lumen Catheters due to Excess Material that May Split or Separate
Medtronic Recalls Neurovascular Products due to Potential Separation and Detachment of
Polytetrafluoroethylene (PTFE) Coating
SentreHeart Recalls FindrWIRZ Guidewire System due to Coating Separation
HeartWare Recalls Ventricular Assist Device Controllers Due to Loose Connector Ports
HeartWare Recalls Ventricular Assist Device Pumps Due to Contamination Causing Electrical Issues
Customer Letter for the Class II Teleflex LMA Mucosal Atomization Devices. (PDF - 76KB)
St. Jude Medical Recalls Implantable Cardioverter Defibrillators (ICD) and Cardiac Resynchronization
Therapy Defibrillators (CRT-D) Due to Premature Battery Depletion
TeleFlex Medical Recalls Tracheostomy Tube Set Due to Possible Disconnection During Patient Use
Leonhard Lang Multi-function Defibrillation Electrodes DF29N Will Not Work with Welch Allyn Automatic
External Defibrillator model AED 10
Baxter Corporation Recalls 50 mm 0.2 Micron Filter Due to the Potential for a Missing Filter Membrane
and Possible Particulate Matter Contamination
DePuy Synthes Recalls Power Tool System Battery Adaptors Due to Possible Explosion Risk
Cook Medical Recalls Roadrunner® UniGlide® Hydrophilic Wire Guide Because of Potential Coating
Contamination
Alere Recalls INRatio® and INRatio2® PT/INR Monitoring System Due to Incorrect Test Results
BioMerieux SA Alerts Customers about Potential Inaccurate Test Results When using NucliSENS®
easyMAG® Magnetic Silica for Nucleic Acid Extraction
Dräger Recalls VentStar Oxylog 3000 Pediatric Patient Breathing Circuit Due to Potential Valve Leakage
CareFusion Recalls AVEA Ventilator Due to an Electrical Issue Which May Cause an Unexpected Shutdown
Stryker Sustainability Solutions (formerly Ascent Healthcare Solutions) Recalls Flush Angiographic
Catheter Due to Tip Separation
HeartWare Inc. Extends Recall to Include Batteries Used in the Ventricular Assist Device Due to
Premature Power Depletion
Medtronic Respiratory and Monitoring Solutions Recalls Battery Pack Used on Patient Monitors Due to
Potential Fire Risk
Hummingbird Med Devices Inc. Recalls Hummi Micro-Draw Blood Transfer Device Due to Potential for
Parts to Disconnect
B. Braun Medical Inc. Recalls Dialog+ Hemodialysis Systems Due Defective Conductivity Sensors
Boston Scientific Corporation Recalls Fetch 2 Aspiration Catheter Due to Shaft Breakage
Vascular Solutions Recalls Guardian II Hemostasis Valve Due to Low Pressure Seal Defect
Focus Diagnostics Recalls Laboratory Examination Kits Due to Inaccurate Test Results
Dexcom Inc. Recalls G4 Platinum and G5 Mobile Continuous Glucose Monitoring System Receivers Due to
Audible Alarm Failure
Cook Medical Recalls Central Venous Catheter and Pressure Monitoring Sets and Trays due to Tips that
May Split or Separate
Verathon Inc. Recalls GlideScope Titanium Single-Use Video Laryngoscope Due to Potential Video Feed
Disruption
Arrow International Inc. Recalls Intra-Aortic Balloon Catheter Kits and Percutaneous Insertion Kits Due to
Sheath Separation Issue
Abbott Vascular Recalls MitraClip Clip Delivery System Due to Issue with Delivery System Deployment
Process
Dräger Evita V500 and Babylog VN500 Ventilators - Recall Expanded to Include Optional PS500 Batteries
with New Power Supply Firmware
Cook Medical Expands Recall for Beacon Tip Angiographic Catheters to Include Additional Product Lots
Dräger Medical Inc. Recalls Emergency Transport Ventilators Due to a System Error that may lead to a
Halt in Ventilation Therapy
Arkray Recalls SPOTCHEM II Test Strips Due to Inaccurate Blood Sugar Readings
Thornhill Research Inc. Recalls MOVES Ventilator System Due to Battery Problem
St. Jude Medical Recalls Optisure Dual Coil Defibrillation Leads Due to Damage that May Prevent Patient
Therapy
Brainlab Cranial Image-Guided Surgery (IGS) System - Navigation Inaccuracy
Stryker Fuhrman Pleural and Pneumopericardial Drainage Sets - Catheter May Break During Insertion
Dräger Evita V500 and Babylog VN500 Ventilators - Issue with Optional PS500 Battery Power Supply May
Cause Ventilators to Shut Down Unexpectedly
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Medical devices – avoiding of pitfalls
R&D costs and time to market
Large R&D costs and long time to market are very common for even relatively
simple medical devices. This is partly due to numerous strict regulations
1960:
Within six weeks Earl Bakken modified a metronome circuit from Popular
Electronics into a wearable battery powered pacemaker.
3 years later Bakken's company, Medtronic, was selling implantable
pacemakers for $375 each.
Today, silver-dollar sized pacemakers sell for about $8,000.
Today it takes 10 to 15 years and millions of dollars from the “gleam in the
inventor’s eye” for a product to reach the marketplace.
Wieringa. AVOIDING PITFALLS IN THE ROAD FROM IDEA TO CERTIFIED PRODUCT (AND THE HARSH CLINICAL ENVIRONMENTTHEREAFTER) WHEN INNOVATING MEDICAL
DEVICES. Belgian Day on Biomedical Engineering
Regenerative medicine
Encyclopedia Britanica:
„application of treatments developed to
replace tissues damaged by injury or disease.
These treatments may involve the use of:
- biochemical techniques to induce
tissue regeneration directly at the site of
damage
- or the use of transplantation techniques
employing differentiated cells or stem cells,
either alone or as part of a bioartificial
tissue.“
Nature Journal:
„collection of techniques and technologies that
aim to restore our physiology to something that
resembles its original condition.“
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Biomedical engineering
Nature Journal
„branch of engineering that applies principles and design concepts of
engineering to healthcare.
Biomedical engineers deal with:
- medical devices such as imaging equipment,
- biocompatible materials such as prostheses or therapeutic biologicals, or
- processes such as regenerative tissue growth.“
Tissue Engineering
Nature Journal:
„set of methods that can replace or repair damaged or diseased tissues with
natural, synthetic, or semisynthetic tissue mimics.
These mimics can either be fully functional or will grow into the required
functionality.“
„Tissue engineering is a branch of regenerative medicine, itself a branch of
biomedical engineering.
Tissue engineering and regenerative medicine are concerned with the
replacement or regeneration of cells, tissues (the focus of tissue engineers) or
organs to restore normal biological function.“
Encyclopedia Britanica:
„Tissue engineering, scientific field concerned with the development of
biological substitutes capable of replacing diseased or damaged tissue in
humans.“
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Tissue Engineering
The idea of tissue engineering emerged in 1988.
Decade later when the infamous “Vacanti
mouse”
The ear was actually a mesh of biodegradable
plastic that was moulded into the desired shape,
sprinkled with cartilage cells collected from a cow,
and implanted under the skin of the mouse. The
researchers removed the ‘ears’ after 12 weeks,
and found that some new cartilage had been
generated within the structure.
the reaction was one of excitement and
wonder. “If we can grow a human ear, why
not a kidney, a liver, an eye?”
30 years of tissue engineering, what has been achieved? Daniel Heath, Lecturer, Biomedical Engineering, University of Melbourne
Tissue Engineering
Most succesfull examples:
30 years of tissue engineering, what has been achieved? Daniel Heath, Lecturer, Biomedical Engineering, University of Melbourne
DO - 10.1007/s00402-013-1834-2
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Tissue Engineering
Most succesfull examples:
30 years of tissue engineering, what has been achieved? Daniel Heath, Lecturer, Biomedical Engineering, University of Melbourne
Nature nanotechnology | VOL 6 | JANUARY 2011. Doi: 10.1038/nnano.2010.246
Tissue Engineering
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Nature nanotechnology | VOL 6 | JANUARY 2011. Doi: 10.1038/nnano.2010.246
Tissue Engineering
Lancet 2006; 367: 1241–46. DOI:10.1016/S0140-6736(06)68438-9
Urinary bladder - 2006
Nature | doi:10.1038/news060403-3
Tissue Engineering
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Lancet 2008; 372: 2023–30. DOI:10.1016/S0140-6736(08)61598-6
Trachea –
decellularised
2008
Tissue Engineering
Lancet 2008; 372: 2023–30. DOI:10.1016/S0140-6736(08)61598-6
Trachea -2008
Tissue Engineering
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http://dx.doi.org/10.1016/ S0140-6736(14)60544-4
2014 – nasal cartilage
Tissue Engineering
N Engl j Med 368;212013; DOI: 10.1056/NEJMc1301237
Resorbovatelná dlaha:
Tissue Engineering
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Biomaterials
American National Institute of Health:
„Any substance or combination of substances, other than drugs, synthetic or
natural in origin, which can be used for any period of time, which augments or
replaces partially or totally any tissue, organ or function of the body, in order to
maintain or improve the quality of life of the individual.“
History
32 000 years ago – first sutures
(NATNEWS, 1983, 20(5): 15–7)
1829 – first study of bioreactivity of steels
(H.S.Levert)
1891 – first Hip and Knee Prostheses, but
succesfully developed in period 1968-
1972.
1912 - anastomose (suture) of blood
vessels (Nobel Prize in medicine)
1941 – first study of implantation of
polymers (cellophane) - wrapping for
blood vessels.
1959 - first fully implantable pacemaker
developed by engineer Wilson Greatbatch
and cardiologist W. M. Chardack
1960s - first silicone breast implant
1969 – first implanted a polyurethane
total artificial heart
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Biomaterials science - multidisciplinarity
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
DOI:10.1177/0269215518815215
https://www.thelondonprosthetics.com/
Biocompatibility
Williams Dictionary of Biomaterials (Williams 1999)
„Ability of a material to perform with an appropriate host response in a specific
situation.“
A cement penetration of 3 to 5 mm is
considered optimal for implant fixation.
All patients in our study had the tibial
surfaces prepared with pulsatile lavage
before the tibial baseplate was placed.
Pulsatile lavage allows for better cement
penetration that increases the tensile and
shear strength of the cement-bone
interface and therefore the probability of
successful implantation. Despite
observing these standards, five patients
(6.3%) with short-keeled tibial baseplates
underwent revision surgery as a result of
aseptic loosening. The failure in four of
the five cases was related to the implantcement
interface. Debonding of the
roughened baseplate from the underlying
cement mantle causes wear debris
between the two surfaces. The produced
particulate metal and cement debris lead
to rapid osteolysis and early failure. DOI: 10.1007/s11999-012-2630-y
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Personalized medicine
Nature Journal:
„Personalized medicine is a therapeutic approach involving the use of an
individual’s genetic and epigenetic information to tailor drug therapy or
preventive care.“
Biomedicine
The branch of medicine that deals with the application of the biological sci
ences, especially biochemistry, molecular
biology, and genetics, to the understanding, treatment, and prevention of
disease.
The American Heritage® Medical Dictionary Copyright © 2007, 2004 by Houghton Mifflin Company.
Published by Houghton Mifflin Company.
A highly nonspecific term for a broad field of study which borrows element
s from the history of human
and veterinary medicine, anatomy, physiology, genetics, pathology, zoolog
y, botanical sciences, chemistry, biochemistry,
biology and microbiology. While traditional medicine is concerned with the
direct practical application of medical knowledge,biomedicine looks at its
history and involves itself in new research to push the limits of what medici
ne is able to accomplish.Biomedicine may also refer to a specific type of tre
atment, generally seen as more ‘natural’ than others, and often availablein
a less regulated context.
Segen's Medical Dictionary. © 2012 Farlex, Inc.
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Nanomaterials
Defining what we mean by a nanomaterial is never straightforward:
for some, the size of the material should be a few nanometres,
for others it should be smaller than a few tens of nanometres,
for still others anything less than a micrometre will do,
for some, one dimension at the nanoscale is enough; for others it should be at
least two or even all three.
For different compounds, the properties that distinguish a nanoscale
specimen from its bulk correspondent occur at different sizes.
The transition is rarely abrupt, and the properties evolve from bulk to
nanoscale in a continuous way, so that establishing a threshold size is arbitrary.
The physical and chemical properties of nanomaterials, as well as
environmental and health toxicity, depend on the precise shape and
composition as well as size.
Nature Nanotechnologyvolume14, page193 (2019)
Nanomaterials
REGULATION (EU) 2017/745 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL
nanomaterial’ means a natural, incidental or manufactured material containing
particles in an unbound state or as an aggregate or as an agglomerate and
where, for 50 % or more of the particles in the number size distribution, one or
more external dimensions is in the size range 1-100 nm;
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Bioinspired
Nature Journal:
„Bioinspired materials are synthetic materials whose structure, properties or
function mimic those of natural materials or living matter. „
Examples of bioinspired materials are light-harvesting photonic
materials that mimic photosynthesis, structural composites that imitate the
structure of nacre, and metal actuators inspired by the movements of jellyfish.
Biomimetic
Nature Journal:
„Biomimetics is an interdisciplinary field in which principles from engineering,
chemistry and biology are applied to the synthesis of materials, synthetic
systems or machines that have functions that mimic biological processes.„
DOI: 10.1016/j.mattod.2013.06.005
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Biofabrication
„Biofabrication refers to the combination of cells, biomaterials, and bioactive
factors with advanced fabrication techniques to generate functional tissue
constructs, with a level of complexity exceeding simple 2D or 3D cultures.“
Biomaterials198 (2019) 78–94
https://www.armiusa.org/
Medical device
REGULATION (EU) 2017/745 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL
„Any instrument, apparatus, appliance, software, implant, reagent, material or
other article intended by the manufacturer to be used, alone or in combination,
for human beings for one or more of the following specific medical purposes:
- diagnosis, prevention, monitoring, prediction, prognosis, treatment or
alleviation of disease,
- diagnosis, monitoring, treatment, alleviation of, or compensation for, an
injury or disability,
- investigation, replacement or modification of the anatomy or of a
physiological or pathological process or state,
- providing information by means of in vitro examination of specimens
derived from the human body, including organ, blood and tissue donations,
and which does not achieve its principal intended action by pharmacological,
immunological or metabolic means, in or on the human body, but which may be
assisted in its function by such means.“
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Medical device – custom made
REGULATION (EU) 2017/745 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL
„Any device specifically made in accordance with a written prescription of any
person authorised by national law by virtue of that person's professional
qualifications which gives, under that person's responsibility, specific design
characteristics, and is intended for the sole use of a particular patient
exclusively to meet their individual conditions and needs.“
However, mass-produced devices which need to be adapted to meet the specific
requirements of any professional user and devices which are mass-produced by
means of industrial manufacturing processes in accordance with the written
prescriptions of any authorised person shall not be considered to be custommade
devices;
LaserImplants™
LaserImplantsare metal 3D printed craniomaxillofacial(CMF) patient specific
implants (PSIs) and surgical guides which can be provided with optional
polycarbonate anatomical models.
Medical device
REGULATION (EU) 2017/745 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL
„clinical evaluation means a systematic and planned process to continuously generate,
collect, analyse and assess the clinical data pertaining to a device in order to verify the
safety and performance, including clinical benefits, of the device when used as intended by
the manufacturer“
„clinical investigation means any systematic investigation involving one or more human
subjects, undertake n to assess the safety or performance of a device“
„clinical evidence means clinical data and clinical evaluation results pertaining to a device
of a sufficient amount and quality to allow a qualified assessment of whether the device is
safe and achieves the intended clinical benefit(s), when used as intended by the
manufacturer“
„clinical performance means the ability of a device, resulting from any direct or indirect
medical effects which stem from its technical or functional characteristics, including
diagnostic characteristics, to achieve its intended purpose as claimed by the
manufacturer, thereby leading to a clinical benefit for patients, when used as intended by
the manufacturer“
„clinical benefit means the positive impact of a device on the health of an individual,
expressed in terms of a meaningful, measurable, patient-relevant clinical outcome(s),
including outcome(s) related to diagnosis, or a positive impact on patient management or
public health“
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Cosmetics
REGULATION (EC) No 1223/2009 OF THE EUROPEAN PARLIAMENT AND OF
THE COUNCIL
„cosmetic product means any substance or mixture intended to be placed in
contact with the external parts of the human body (epidermis, hair system,
nails, lips and external genital organs) or with the teeth and the mucous
membranes of the oral cavity with a view exclusively or mainly to cleaning
them, perfuming them, changing their appearance, protecting them, keeping
them in good condition or correcting body odours“
„substance means a chemical element and its compounds in the natural state or
obtained by any manufacturing process, including any additive necessary to
preserve its stability and any impurity deriving from the process used but
excluding any solvent which may be separated without affecting the stability of
the substance or changing its composition“
„mixture means a mixture or solution composed of two or more substances“
Bioinspired structural materials
Materials-design problem - the two key structural properties (strength and
toughness) tend to be mutually exclusive:
strong materials are invariably brittle,
tough materials are frequently weak.
Bioinspiration
Highly mineralized, mostly ceramic, natural structures, such as tooth minimize
wear and provide protection.
A unique aspect of these materials is that they utilize different structures or
structural orientations:
Tooth
hard surface layers so as to resist wear and/or penetration,
tough subsurface to accommodate the increased deformation.
DOI: 10.1038/NMAT4089
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Bioinspired structural materials
Tooth enamel is remarkably well conserved
throughout the natural world.
Vertical columns of stiff, inorganic material stand
embedded in a polymer matrix.
The energy from the vibrations the tooth is subjected
to during chewing is dissipated thanks to friction, as
the flexible polymer pillars of the tooth oscillate more
than the stiffer ones.
Natural materials often combine stiff and soft
components in hierarchical structures.
The structure of the enamel
Humans
Tyrannosaurus rex
Syntetic
DOI: 10.1038/nature21410
Bioinspired structural materials
Natural structural materials are built at ambient temperature from a fairly
limited selection of components.
Limited number of components that have relatively poor intrinsic properties.
Superior traits stem from naturally complex architectures that encompass
multiple length scales.
They usually comprise hard and soft phases arranged in complex
hierarchical architectures, with characteristic dimensions spanning from
the nanoscale to the macroscale.
The resulting materials are lightweight and often display unique combinations
of strength and toughness, but have proven difficult to mimic synthetically.
Any rational strategy must incorporate nano-, micro- and macroscale features,
and thus involve the so-called mesoscale approach.
DOI: 10.1038/NMAT4089
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Structural hierarchy of the gecko adhesive system
Kellar Autumn, and Nick Gravish Phil. Trans. R. Soc. A 2008;366:1575-1590
Bioinspired structural materials
In the composites, the interface are crucial:
For instance, the enamel/dentin interface combines compositional gradients
with scalloped interfaces, which ensures stability.
DOI: 10.1038/NMAT4089
doi.org/10.1590/S0103-64402006000300001
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Bioinspired structural materials
At the microscopic level, natural composites are usually complex and
anisotropic.
They can have layered, columnar or fibrous motifs.
Quite often, the same structure can exhibit distinct layers with different
motifs.
These motifs are usually orchestrated in sophisticated patterns, such as
columns of circular layers in bone.
DOI: 10.1038/NMAT4089
Nature Materialsvolume15, pages1195–1202 (2016)
Bulk vs Surface
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Bulk vs Surface – titanium surfaces – hip replacement
SEM images of different titanium surfaces at magnification of 8000×.
(a) Polished titanium surface.
(b) Spear-type surface showing a dense nanostructure comprising short and thin spears. The inset
shows a SEM image of a nano-spear with a cone-shaped cap taken at 100,000×magnification.
(c) Pocket-type surface formed by intertwining of the longer and wider nano-spears.
ScientificReports 8, Article number: 1071 (2018)
Bulk vs Surface – titanium surfaces – hip replacement
Adherence of S. epidermidis on different
titanium surfaces after 2 hours’
incubation. (a) Representative CLSM
images with LIVE/DEAD staining of S.
epidermidis on polished, spear-type and
pocket-type titanium surfaces (left to
right). (b) Different contact mechanisms
of S. epidermidis on different titanium
surfaces as visualized by SEM (Images
were taken at 40° tilt with magnification
of 25000×). (b1) S. epidermidis cells
rested on the polished titanium surfaces;
(b2) for spear-type surfaces, S.
epidermidis cells settled on the top of
spears resulting in point contacts; (b3)
for pocket-type surfaces, S.
epidermidissettled inside the pocket-like
nanostructures (outlines of selected
pockets are marked by dashed red lines).
The longer, intertwined nano-spears
were also observed to provide
colonization sites for bacteria (arrow). (c)
Surface area covered by both live and
dead bacteria in the field of view for each
titanium surface. (d) Bactericidal
efficiency of the different titanium
surfaces, as indicated by the proportions
of dead cells among the adherent
bacteria. *Statistically significant
difference (p < 0.05). Three independent
experiments were performed for each
substrate type.
ScientificReports 8, Article number: 1071 (2018)
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Bulk: Stres (napětí) and strain (deformace)
Stress (napětí) is defined as the force experienced by the object which
causes a change in the object while a strain (deformace, přetvoření,
veličina – nezaměňovat s „deformation“ – jakožto jevem, procesem ) is
defined as the change in the shape of an object when stress is
applied. Stress is measurable and has a unit (n/m2) while a strain is a
dimensionless quantity and has no unit.
Compression (tlak) stress
Tensile (tah) stress
Shear (smyk) stress
Torsion (kroucení) stress
DO - 10.1088/1742-6596/908/1/012019
doi.org/10.1007/978-981-32-9971-9_28
Bulk: Stress / strain curve
HOOKE’S LAW F = kΔL, where ΔL is the amount of deformation (the change
in length, for example) produced by the force F, and k is a proportionality
constant that depends on the shape and composition of the object and the
direction of the force ΔL=
https://courses.lumenlearning.com/physics/chapter/5-3-elasticity-stress-and-strain/
A graph of deformation ΔL versus applied
force F.
The straight segment is the linear region
where Hooke’s law is obeyed.
The slope of the straight region is 1/k.
For larger forces, the graph is curved but
the deformation is still elastic—ΔL will
return to zero if the force is removed.
Still greater forces permanently deform
the object until it finally fractures.
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Bulk: Toughness (houževnatost)
Measures the energy required to crack a material.
Materials can be:
Ductile (tažný) — deforms before it breaks
Brittle (křehký) — breaks before it deforms
Brittle materials break often without warning. They have little elasticity
and do not stretch easily.
Brittle vs. Ductile fracture (a) Very ductile, soft
metals (e.g. Pb, Au) at room temperature, other
metals, polymers, glasses at high temperature.
(b) Moderately ductile fracture, typical metals (c)
Brittle fracture, cold metals and ceramics.
DOI - 10.5772/18127
Brittle
Bulk: Strength (pevnost)
Strength of a material is its ability to withstand an applied stress without
failure or plastic deformation
Strong
Weak
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Bulk: Strength (pevnost) vsToughness (houževnatost)
Strength measures the resistance
of a material to failure, given by the
applied stress (or load per unit
area)
Toughness measures the energy
required to crack a material; it is
important for things which suffer
impact
Increasing strength usually leads to
decreased toughness
http://www-materials.eng.cam.ac.uk/
Bulk: Stiffness (tuhost) vs flexibility (pružnost)
Stiffness is the extent to which an object resists deformation in response
to an applied force.
The complementary concept is flexibility: the more flexible an object is,
the less stiff it is.
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Bulk: Young's Modulus - Density
'Stiffness' measures how much
something stretches when a load is
applied. Young's modulus measures
stiffness and is a material constant,
i.e. it is the same whatever the size
of the test-piece.
Many applications
require stiff materials, e.g. roof
beams, bicycle frames - these
materials lie at the top of the chart
Many applications require low
density materials, e.g. packaging
foams - these materials lie to
the left of the chart.
Stiff lightweight materials are hard
to find - composites appear to offer
a good compromise, but they are
usually quite expensive.
http://www-materials.eng.cam.ac.uk/
Bulk: Strength - Density
Strength measures the resistance of a material to failure, given by the
applied stress (or load per unit area)
http://www-materials.eng.cam.ac.uk/
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Bulk: Strength - Elongation
Strength measures the
resistance of a material to
failure, given by the
applied stress (or load per
unit area)
Elongation measures the
percentage change in
length before fracture
Elongation to failure is a
measure of ductility
http://www-materials.eng.cam.ac.uk/
Bulk: Architecture
Figure 2: Ultramicrographs by SEM of different tissue engineered based scaffolds.
(A and B) are porous scaffolds. These types of scaffolds are porous in nature and their porosity is high. The pore size is usually between
10 to 100 μm but it may be larger. No fiber structure is visible in these scaffolds and the molecules are not arranged as fibers. The
density of the pores is more than 50% in these types of scaffolds.
(C to F) are fibrous or fiber based scaffolds. (C) is an amorphous scaffold which has some porosity between its fibers while (D) is a
highly aligned scaffold which has lower porosity than (C).
(E) is a unique highly aligned scaffold but the fibers have been polymerized and aligned in two different directions so that some of the
fibers are perpendicular to others. This unique configuration of the fibers has produced large pores between the fibers.
(F) is a moderately aligned scaffold in which the collagen fibers are mostly aligned but an acceptable porosity exists between the
fibers.
(G) is a fibrous scaffold which have been cross-linked so that the fibers have been fused together but some porosity is still preserved in
the scaffold. (H) is a hybrid scaffold in which both the micro and nano structured fibers are present in the scaffold. Scale bar for A: 1
μm, B: 500 nm, C to F: 1 μm, G: 2 μm, H: 2.4 μm
https://www.omicsonline.org/articles-images/2161-0673-3-126-g002.html
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Surfaces: Chemistry
Surfaces can be composed of different
chemistries:
- Atomic.
- Supramolecular (entities of greater
complexity than individual
molecules — assemblies of
molecules that bond and organize
through intermolecular
interactions).
- Macromolecular (very large
molecule, as a colloidal particle,
protein, or especially a polymer,
composed of hundreds or
thousands of atoms).
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
Surfaces: Topography
Rough.
Stepped.
Smooth.
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
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Surfaces: Topography
Wrzecionko et al. 2017. DOI: 10.1021/acsami.6b15774
Surfaces: Topography
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Surfaces: Homogeneity
Structurally or compositionally
inhomogeneous in the plane of
the surface such as phaseseparated
domains or
microcontact printed lanes.
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
Biomaterials science : an introduction to materials in medicine.ISBN 0-12-582463-7
Surfaces: Homogeneity
Inhomogeneous with depth into
the specimen or simply
overlayered with a thin film.
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
07.06.2021
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Highly crystalline or disordered
Biomaterials science : an introduction to materials in medicine. ISBN 0-12-582463-7
Surfaces: Isotropy
DOI: 10.5194/esurfd-3-1399-2015
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Acta Biomaterialia(2010) doi:10.1016/j.actbio.2010.01.016
Hydrofilicity
Protein–surface interactions
Cellular responses to materials in a biological medium reflect greatly the
adsorbed biomolecular layer.
Diffusion and binding kinetics underlie the
‘‘Vroman effect“, which describes the
exchange of proteins adsorbed on surfaces.
Earlier adsorbed proteins (mostly low MW)
by other proteins with stronger binding
affinities
doi.org/10.1016/j.bios.2008.07.036
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Protein–surface interactions
Topographic atomic force microscopy images that
illustrate the structural evolution of an adsorbed
cellulase layer on polystyrene as a function of
incubation time (shown in the figure labels).
Large aggregates and unaggregated proteins are
observed at 1 min (top-left).
The aggregates reduce in size at 20 min (top-right)
and, at 60 min (middle), they form a taller and less
dense network structure.
At 3 h (bottom-right), protein aggregates are
discernible in a denser layer of reduced height.
At 24 h (bottom-left), the adsorbed proteins have
formed taller rod-like structures. The average height
and root mean square (RMS) roughness are given for
each image.
http://dx.doi.org/10.1016/j.colsurfb.2012.10.039
Protein–surface interactions
AFM views of non-specific protein adsorption to a base plastic material (naked polypropylene slides) (A)
and the surface after coating with PC-methacrylic copolymer (B).
Silds were exposed to pure water (1; negative control), or 10nM aqueous solutions of TGF-β (2; a
hydrophobic protein), histone H1 (3; a basic protein), and erythropoietin (4; a glycoprotein hormone) as
typical model proteins. After incubating with the solution for 1h at 37°C, the slides were washed three
times with 0.05% (w/v) Tween 20 in deionized water and then air-dried.
DOI: 10.1016/j.bbagen.2013.11.009
Polypropylene - hydrophobic
Polypropylene coated with PC-methacrylic copolymer
pure water
pure water
hydrophobic protein
hydrophobic protein
glycoprotein
glycoprotein
histone H1
histone H1
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Protein–surface interactions
Protein–surface interactions depend on the properties of both the protein
and the surface.
Protein properties:
Size, net charge, stability and unfolding rate regulate the affinity of proteins to
surfaces.
Smaller proteins adsorb faster to surfaces because of faster diffusion.
Larger proteins present a higher surface for interactions with material surfaces, but diffuse
slower and take a longer time for surface adsorption.
Consider in context of mixtures of proteins, such as serum .
Proteins with stronger and more stable interactions replace proteins with low surface affinity.
The overall combination of protein properties is what dictates the stability of
the protein’s adhesion to and stability of interaction with the surface
doi.org/10.1016/j.ymeth.2015.08.005
Protein–surface interactions
Surface properties
Topography, chemical composition, hydrophobicity, heterogeneity and surface
potential affect protein adsorption and adhesion.
Proteins expose hydrophilic amino acids, which are readily available to
interact with material surfaces because physiological conditions involve
aqueous environments.
However, the interactions of hydrophobic surfaces with the hydrophobic
domains of proteins lead to the strongest protein adsorption states.
Protein affinity to charged surfaces is stronger when the pH of the
environment is near the protein’s isoelectric point due to decreased
protein–protein interactions in solution, as proteins assume a neutral net
charge.
The distribution of charged residues on proteins and protein–protein
interactions in solution therefore affect these adsorption events and their
stability easily varies with the pH and ionic state of the culture media.
The rate of protein unfolding accelerates protein–material interactions.
Yet unfolding can also lead to protein denaturing and loss of protein
stability.
doi.org/10.1016/j.ymeth.2015.08.005
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Protein–surface interactions
doi.org/10.1016/j.ymeth.2015.08.005  Philos. Trans. A: Math. Phys. Eng. Sci. 370
(2012) 2321–2347  J. Mater. Chem. 17 (2007) 943–951.
Protein–surface interactions
Processes for the change in composition of a layer adsorbed from a mixture solution
by exchange of earlier adsorbed proteins with other proteins.
(A) Initially adsorbed protein 1 (blue) desorbs, leaving a vacancy for protein 2 (red)
to adsorb.
(B) Initially adsorbed protein 1 is displaced by protein 2 which has a stronger binding
affinity to the surface.
(C) Protein 2 embeds itself in previously adsorbed protein 1 to form a transient
complex (top); the complex then turns, exposing protein 1 to solution (middle);
protein 1 desorbs into the solution and protein 2 remains on the surface (bottom).
http://dx.doi.org/10.1016/j.colsurfb.2012.10.039
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Protein–surface interactions
For example:
Hydrophobic surfaces (e.g., Teflon) with low surface energy denature proteins.
Very hydrophilic surfaces (e.g. polyacrylamide hydrogels) sterically hinders
protein adsorption.
Hydrophobic surfaces with low surface energy (e.g. PDMS Polydimethylsiloxane)
promotes weak interactions with proteins.
Protocols to attach proteins to these materials first modify the undesirable
material surface properties and then functionalize the material surface to
promote stable and strong protein attachment using polar interactions,
hydrophobic interactions, ionic bonds, and/or covalent bonds
Of these, covalent bonds are the strongest and least dependent on the
electrochemistry of the extracellular environment
doi.org/10.1016/j.ymeth.2015.08.005
Proteins
The protein interface is a mediator of cell/material interactions.
ECM proteins, adsorbed onto the material surfaces after implantation in
vivo and from the culture media in vitro, are recognised by cells through
integrins.
Initial interaction leads to:
integrin clustering,
internal recruitment of cytoplasm proteins,
forming focal adhesions and mediating cell adhesion and contractility.
Physicochemical properties (chemistry, topography and mechanics) play an
important role on the adsorption of proteins onto the material surface and
influence:
protein surface density,
Protein conformation,
Protein distribution.
doi.org/10.1016/j.actbio.2018.07.016
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Proteins
Above mentioned directing cell response:
early events of cell attachment and spreading,
controlling later events such as proliferation, matrix reorganization and
differentiation.
The mechanical organization and reorganization of ECM proteins is
important physiological event that happens after the initial cell/protein
interaction.
In vivo cells secrete and reorganize proteins into fibrils to form their own ECM,
which provides them with mechanical support and local growth factors
delivery.
doi.org/10.1016/j.actbio.2018.07.016
ECM
Cell interactions with the ECM are highly dynamic in vivo:
Cells receive information from specific cues in the ECM,
Respond to these inputs by remodeling the surrounding matrix and/or
secreting new components.
Proteolytic degradation for the removal of excess ECM:
Mostly active during development, wound healing and regeneration of tissues.
When misregulated, can contribute to diseases such as fibrosis, arthritis and
cancer invasion.
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Fibronectin
One of ECM protein is fibronectin (FN) that plays a key role in cell adhesion
and proliferation, controls the availability of growth factors, and so
contributes to cell differentiation.
FN is synthesized by various anchorage-dependent cells, which then assemble it
into a fibrillar network through an integrin dependent mechanism.
FN assembly is the initial step which orchestrates the assembly of further ECM
proteins such as collagen.
doi.org/10.1016/j.actbio.2018.07.016
Fibronectin – degradation
doi.org/10.1016/j.actbio.2018.07.016
Cell-mediated FN matrix degradation on PEA surfaces coated with FN at 20 mg/ml for 1 h. a) Cell-mediated FN
degradation at the nanoscale. AFM phase images show different protein patterns in dependence of the proximity (far
vs around) to cells. FN distributions in areas around cells and far from cells are showed at higher magnification in b).
07.06.2021
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Protein matrix degradation during increasing culture time on FN-coated PEA substrates. Cells start degrading the
FN layer from short culture times. Scale bar: 25 mm.
Fibronectin – degradation
doi.org/10.1016/j.actbio.2018.07.016
Cell-mediated FN reorganization on FN-coated PEA after 30 min and 2 h incubation. FN degradation is observed
regardless of the culture time (a-b, d-e), FN concentration (a-b) and adsorption time (d-e). However, less
evidences of degradation are observed with higher FN concentration, which results in better spread cells. In the
presence of serum (c,f) no signs of FN degradation are observed and cells present a spread morphology. Top scale
bar: 50 mm, bottom scale bar: 25 mm.
Fibronectin – degradation
doi.org/10.1016/j.actbio.2018.07.016
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Differentiation and nanotopography geometries
Three basic nanotopography geometries include nanogrooves (a),
nanopost array (b), and nanopit array (c).
The speculative pathways (d) for cell-shape-directed osteogenic and
adipogenic differentiations of MSCs were examined in growth medium.
RhoA, Ras homolog gene family member A; ROCK, Rho-associated protein
kinase.
https://doi.org/10.1038/boneres.2015.29
TopoChip design.
Hemant V. Unadkat et al. PNAS 2011;108:16565-16570
©2011 by National Academy of Sciences
Aplikace High-throughput technologie pro „screening“ biologických vlastností biomateriálů.
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TopoChip fabrication and characterization.
Hemant V. Unadkat et al. PNAS 2011;108:16565-16570
©2011 by National Academy of Sciences
Topografie - vliv na morfologii
TopoChip tvořený jedním materiálem poly(DL-lactic acid).
2 176 různých povrchových topografií.
Primary human mesenchymal stromal cells (hMSCs) byly vysazeny na
povrchy.
Po 8 hodinách fixace a barvení aktinu.
doi/10.1073/pnas.1109861108
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Morphology of hMSCs on different TopoUnits.
Hemant V. Unadkat et al. PNAS 2011;108:16565-16570
©2011 by National Academy of Sciences
Topografie – vliv na proliferaci
Synchronizace buněčného cyklu – deprivace séra.
Přidání séra + analogu nukleotidu (EdU).
8 hodin kultivace.
Barvení DNA.
doi/10.1073/pnas.1109861108
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Cell proliferation assay.
Hemant V. Unadkat et al. PNAS 2011;108:16565-16570
©2011 by National Academy of Sciences
Topografie - diferenciace
Kultivace bez přítomnosti růstových faktorů.
Alkaline phosphatase – marker rané osteogenní diferenciace.
doi/10.1073/pnas.1109861108
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TopoChipy s dalšími materiály
doi/10.1073/pnas.1109861108
Topography & hydrophilicty
DOI: 10.1021/acsami.6b15774
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Acta Biomaterialia 9 (2013) 7014–7024. doi:10.1016/j.actbio.2013.02.039
TCP/HA
TCP/HA
Sr–HT–gahnite
Sr–HT–gahnite
Biointerfaces – TOPOGRAPHY + COMPOSITION
Isotropy - biomimetic
07.06.2021
46
DOI: 10.5772/21197
Isotropy – fiber alignment
Isotropy – fiber alignment
Adhesion morphology and alignment of human mesenchymal stem cells (hMSCs) on original (left) and stretched (right)
PCL-9.2 nanofibers. hMSCs on each fiber type were stained with f-actin (red) and nucleus (blue). hMSCs were cultured
at 37 °C for 24 h. Large scan (left) and magnified (right) images show global and local alignment of hMSCs.
https://doi.org/10.3390/fib7030020
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Isotropy
doi:10.1155/2012/797410
Isotropy
doi:10.1155/2012/797410
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Elektricky vodivé polymery – motivace ke studiu
Rune Elmqvist 1957 – první plně implantovatelný kardiostimulátor
Cochleární implantát – nejčastěji užívané biologické rozhraní s elektrogenní
tkání
2016 – „cardiac patch“ umožňující online stimulaci a snímání odezvy při hojení
infarktu
www.medmuseum.siemens-
healthineers.com/
DOI: 10.1186/1471-2482-
13-S2-S1
DOI: 10.1038/NMAT4590
Elektricky vodivé polymery – motivace ke studiu
Vliv elektrického pole na eukaryotické buňky – přestavba cytoskeletu DOI: 10.1016/j.proghi.2008.07.001
5h, 0 V/cm 5h, 5 V/cm
Směr el. pole
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Elektricky vodivé polymery
Biomaterials (2006) 27(3):473-84.
Traction forces
Cells generally apply traction forces to the networks or the surfaces to
which they are bound
Cell traction forces is defined as a tangential tension exerted by cells to the
ECM or the underlaying layer. It is generated by actomyosin interaction and
actin polymerization and regulated by intracellular proteins.
Traction microscopy image: Traction stress field exerted by a
rat pulmonary microvascular endothelial cell upon its
substrate. Inset: Phase contrast image at reduced
magnification. Scale: Shear stress in Pascals.doi:10.1007/s10439-009-9661-x
Am J Physiol Cell Physiol 2005;289(3):C521–C530.
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Micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal
contractility and stem cell differentiation. Furthermore, early changes in
cytoskeletal contractility predicted later stem cell fate decisions in single
cells.
doi:10.1038/nmeth.1487
Traction forces - substrate „nano-rigidity“
Funkční podjednotky tkání
Jedná se o nejmenší jednotky, které zajišťují funkci tkáně či orgánu.
Funkční jednotky jsou v řádech ~100 mm.
Každý orgán je tvořen 10-100 x 106 funkčními jednotkami.
Každá funkční jednotka je tvořena rozdílnými typy buněk a extrucelulární matrix.
Rozdělení funkční jednotky na menší části (buňky a ECM) dochází ke ztrátě funkce tkáně
či orgánu.
Obě části, tedy buňky i ECM jsou nenahraditelné.
Funční jednotka je tedy minimální hladina na které TE pracuje.
Mikro-environment tkáně je dán:
Hustota buněčného osazení (Cellularity)
Buněčná komunikace (Cellular Communications)
Přítomnost živin (Local Chemical Environment)
Geometrie tkáně (Local Geometry)
Nature nanotechnology | VOL 6 | JANUARY 2011. Doi: 10.1038/nnano.2010.246
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Tkáňové inženýrství (Tissue Engineering)
Tkáňové inženýrství kombinuje principy a metody inženýrských a
biologických věd k vývoji biologických náhrad určených k obnově, udržení či
zlepšení funkce tkání (či orgánů).
Nejběžnější způsob je založen na imobilizaci vhodných buněk v porézních,
biodegradibilních a biokompatibilních scaffoldech poskytujících templát k
vývoji tkáně a následné kultivaci.
Klíčovými body jsou:
Selekce vhodných buněčných linií
Příprava vhodného scaffoldu vytvářejícímu prostor pro buněčné procesy.
Vytvoření vhodných podmínek pro kultivaci buněk v 3D struktuře vedoucí k
proliferaci a diferenciaci buněk v cílovou tkáň - bioreaktory.
Jsou dobře zvládnuté techniky pro kultivaci 2D struktur:
Kožní náhrady po vředech či popáleninách.
Kultivace v 3D struktuře je však velmi odlišná od 2D např.:
Nutnost zapojení více buněčných typů.
Diferenciace.
Dynamičnost procesu – neustále se měnící biologické, fyzikální či
mechanické prostředí v in vivo podmínkách.
Navíc se vše mění v čase – jedná se tedy spíše o 4D.
Kost Cévy
Chrupavka
BIOREAKTORY.
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Bioreaktory - Úvod
Lidské buňky jsou velmi náchylné ke změně podmínek prostředí např.
kumulace toxických produktů metabolismu, jež jsou v tradičních,
statických, kultivačních technikách běžné.
In vivo je dynamický systém vytvářející komplexní síť vzájemných interakcí
buňka-buňka, buňka-prostředí.
Bioreaktory slouží k co nejvěrnější simulaci in vivo podmínek.
Výběr parametrů jež jsou sledovány je dán cílem kultivace.
Množení buněk pro transplantaci – hematopoetické buňky, kmenové
buňky, krevní buňky.
3D struktury pro implantace.
Orgány.
Bioreaktory zajišťují také možnost studia vlivu různých mechanických,
biochemických a dalších stimulů v 3D struktuře tkáně za in vitro podmínek.
Bioreaktory - fermentory
Bioreaktor je obecný pojem používaný pro uzavřené systémy kultivace
umožňující kontrolu jednoho či více faktorů ovlivňujících biologické
pochody.
V průmyslovém biotechnologickém využití se jedná především o tzv.
fermentory.
Umožňují růst eukaryotických či prokaryotických buněk ve vysoké hustotě
zajišťující vysokou produkci metabolických produktů, enzymů či overexpresi
rekombinantních genových produktů.
Patří sem:
Stirred tank reactors.
Packed beds.
Membrane bioreactors.
Kultivují se monokultury
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Bioreaktory pro TE
V tkáňovém inženýrství jsou bioreaktory používány k zajištění
kontrolované a reprodukovatelné buněčné proliferace.
Kontrolují se:
Teplota
pH
Koncentrace plynů
Míra toku médií
Tlak
Hydrodynamické a mechanické síly
Rozdíl oproti jiným kultivačním technikám je v nutnosti zajistit aby buňky
proliferovali a diferencovali stejně jako v podmínkách in vivo.
Oproti fermentorům je navíc často požadavek na kultivaci více linií buněk.
Náročnost se zvyšuje také nutností inkorporace vhodného scaffoldu.
Cílem tedy není produkce určitého objemu buněk či produktu
metabolismu, ale růst mnoha buněčných linií v organizované 3D
struktuře.
Obecné požadavky na design bioreaktorů pro TE
Základní podmínky jež by měl splňovat:
Vytváření vhodných podmínek srovnatelných s podmínkami in vivo (zajištění
proliferace a diferenciace).
Uniformní rozložení buněk v 3D scaffoldu.
Udržování optimální koncentrace živin v celém prostoru.
Vytváření vhodných fyzikálních stimulů.
Zajištění sterility prostředí (zajištění sterilizace pomůcek, médií, kultivačních
technik a s ohledem na délku inkubace také možnost sterilní výměny médií a
odběru vzorků).
Možnost změny podmínek v čase.
Splnění Good Manufacturing Practice a Quality Asurance.
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„Osazení“ bioreaktoru
Pro úspěšnou kultivaci je nezbytná maximální homogenita osazení
scaffoldu buňkami.
Čím je vyšší hustota buněčného osazení na začátku tím je lepší a uniformní
tvorba tkáně.
To může být problematické i u malých scaffoldů a s jejich velikostí se tento
problém zvyšuje.
Existují dvě možnosti:
Statický systém - přichycení buněk na scaffold a následné přenesení do
bioreaktoru.
Dynamický systém - okolo statického scaffoldu omýváno médium obsahující
buňky.
Efektivní pro dosažení vysoké hustoty a zároveň uniformity.
Vede však k horšímu osazení uvnitř scaffoldu.
Perfusní dynamický systém kdy je opakovaně protlačováno médium skrz
scaffold.
Nejúčinnější systém.
Transport živin
In vivo jsou tkáně obvykle vzdáleny do 100µm od kapiláry dodávající živiny.
Z toho by se mělo vycházet při přípravě struktur zajišťujících mikroenvironment
v rámci jednotlivých podjednotek tkáně.
Subjednotka by tak měla představovat kostku o straně 100µm což
představuje cca 500 – 1000 buněk (chrupavka má jen 1 buňku na 100 µm3).
Pokud je cílem tkáň o větších rozměrech pak jsou buňky vzdálené více než
100µm vystaveny nedostatečnému zásobování živinami a odvodem
odpadních látek. Důsledkem je vznik hypoxických a nekrotických center.
In vivo je tento problém řešen pomocí vlásečnic.
In vitro musí být tento problém řešen konstrukcí scaffoldu (porositou) a
bioreaktoru (tokem média).
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Vnitřní struktura scaffoldu
Poréznost ovlivňuje dostupnost živin, odvod odpadních látek a regulačních
molekul, mezibuněčnou komunikaci, prostorový růst buněk, fyzikální stres.
Vzdálenost - schopnost difuze látek je odvislá od vzdálenosti - buňky bez
kultivace v bioreaktorech dokáží růst jen cca do 400 μm od povrchu (v závislosti
na porositě).
Koncentrace - látky musí být k dispozici ve fyziologické koncentraci (přívod a
odvod musí odpovídat jejich spotřebě či produkci) aby efekt nebyl inhibiční až
toxický.
Homogenita - koncentrace látek by měla být stejná v různých místech – tok
tekutiny by tedy měl být všude vyrovnaný.
Koncentrační gradient – u některých látek je třeba vytvářet koncentrační
gradient s ohledem na jejich funkci (může být řešeno vazbou v scaffoldu).
Transport živin
Živiny a plyny jsou transportovány pomocí:
Toku živného média.
Difuzí.
Rychlost toku média: in vivo je absorpce kyslíku 25 – 250 µmol 02/cm3/h a
průtok tekutiny (pefusion rate) 0,07 ml/cm3/min což je založeno na
průměrné koncentraci buněk 500 milionů buněk na cm3.
Kyslík
Fyziologická koncentrace ~ 0.2 mM
Spotřeba 0.05 – 1 mmol/106 buněk/hodinu
Glukoza a další primární metabolity
Spotřeba 0.05 – 1 mmol/106 buňěk/hodinu
Amino kyseliny, růstové faktory
Fyziologická koncentrace v řádech nM – mM
Spotřeba 0.01 – 1.0 nmol/106 buněk/hodinu
Odpadní metabolity – kysleina mléčná, amoniak
Odvod 0.01 – 0.2 mmol/106 buněk/hodinu
Průtok by tedy měl být zajištěn zhruba 50 – 400 mL/min/106 buněk.
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Transport živin
Příklad kultivace bovinních chondrocytů na scaffoldu z poly(glycolic)acid.
Chondrocyty produkují glycosaminoglycany jež jsou barveny červeně.
Biochemechanické stimuly
Tkáně mají v in vivo systémech také mechanické funkce (kosti, chrupavky,
vazivo, cévy).
Působením vhodných mechanických vlivů při in vitro kultivaci dochází k
tvorbě struktur podobných in vivo.
Dochází tedy k ovlivnění jak diferenciace tak proliferace a morfologie buněk a
ECM.
Stres daný dynamickým tlakem toku tekutiny (Fluid dynamic stress, or
‘‘shear stress’’) je považován za klíčový parametr vedoucí k aktivaci
signálních drah podstatných pro mechanické vlastnosti tkáně.
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Biomechanické stimuly
Každá tkáň vyžaduje odlišné další mechanické stimuly:
Pulsující radiální stres tubulárního scaffoldu osazeného hladkosvalovými
buňkami zlepšuje strukturní organizaci umělých cév. Dále zvyšuje produkci
elastinu čímž zlepšuje mechanické vlastnosti graftu.
Dynamická deformace stimuluje syntézu glycosaminoglykanů chondrocyty a
zlepšuje tak mechanické vlastnosti chrupavky.
Rotační tlaky mesenchymálních progenitorových buněk v kolagenové matrici
vedou k spojování buněk a formaci orientovaných kolagenových vláken.
Mechanické stlačování a cyklický hydrostatický tlak ovlivňuje významně
genovou expresi a produkci extra celulární matrix.
Vliv na chrupavku in vivo i in vitro v kultuře chondrocytů je ovlivňována:
Komprese mění buněčnou proliferaci, metabolismus pojivové tkáně a obsah
pojiva.
Statický tlak způsobuje trvalou deformaci tkáně zároveň inhibuje syntézu pojiva.
Dynamický tlak o určité amplitudě a frekvenci způsobující tok tekutiny v tkáni a
její deformaci stimuluje syntézu pojiva.
Typy bioreaktorů
Existuje celá řada různých typů. V následující části budou uvedeny jen
vybrané příklady.
Uvedené výhody a nevýhody jsou obecné a vždy závisí na konkrétní tkáni
jež má být generována a scaffoldu.
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In vivo bioreaktory
Využívají in vivo tkáně jako zdroj živin a růstových faktorů.
Scaffold je umístěn do tkáně a následně jej osadí buňky vlastního těla
(především krevní elementy a SC).
Problém je s nalezením vhodných scaffoldů a jejich povrchových úprav tak
aby stimulovali buňky k proliferaci a diferenciaci správným směrem.
Spinner flasks
Buňky jsou transportovány na povrch i do scaffoldu pomocí konvekce.
Míchání média zvyšuje povrchový transfer média a podporuje růst buněk
nicméně generuje turbulence které mohou mít negativní dopad na tvorbu
tkáně.
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Rotating-wall vessels
Poskytuje dynamické prostředí při vysoké dostupnosti živin a odvodu
odpadních látek.
Vytváří nízký smykový stres.
Jsou velmi vhodné pro tvorbu chrupavky protože indukují chondrogenezi.
Hollow-fiber bioreactors
Využívány k vytvoření dostatečné proliferace u buněk s intenzivním
metabolismem (hepatocyty).
Jsou dvě možnosti uspořádání:
Buňky jsou umístěny do gelu uvnitř dutých vláken (o průměru cca 200µm) a
médium je protlačováno skrze stěny vláken.
Buňky jsou v prostoru mezi vlákny a médium je tlačeno skrze vlákna.
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Direct perfusion bioreactors
Médium je tlačeno skrz póry scaffoldu.
Využíváno pro osazení i následnou kultivaci.
Při osazování jsou buňky transportovány skrze póry přímo do scaffoldu což
zajišťuje vysokou uniformitu.
V průběhu kultivace tok zajišťuje vysokou tvorbu buněčné hmoty.
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Schematics of an undisturbed cell, cells under shear stress,
pressure, and their gradients
DOI: 10.1007/s10544-011-9543-5
Cyclic uniaxial stretch and stress fibers
The tension generated by contraction of adherent cells against their
underlying surface results in an internal stress field that depends on the
organization of the cytoskeleton and the associated adhesive contacts.
Intracellular forces have an important role in cellular functions such as
migration, proliferation, apoptosis, differentiation, and gene expression.
Actin stress fibers, which are formed in response to cell contraction,
consist of bundles of actin microfilaments cross-linked by -actinin, myosin,
myosin light-chain, tropomyosin, and other proteins arranged in a manner
similar to that in muscle sarcomeres.
Stress fibers represent the main contractile apparatus in non-muscle cells and
are the primary structures associated with intracellular tension.
Stress fibers terminate at focal adhesions, which attach the cell to the
extracellular matrix.
Isometric contraction of a cell would result in tension development in the
stress fibers, which are anchored at their ends.
Cyclic uniaxial stretch induces the orientation of stress fibers in
endothelial cells (ECs) perpendicular to the principal direction of stretch.
doi10.1073pnas.0506041102
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Stress fibers and Rho pathway
doi10.1073pnas.0506041102
• Top (A) and side (B) views of a stretch chamber and indenter to illustrate the principle of cell
stretching.
• An I-shaped teflon indenter pushed up against a silicone rubber membrane secured to a square
frame results in a principal stretch oriented along the long axis of the indenter.
• The small tension generated in the orthogonal direction is opposed by the tendency for the
membrane to compress orthogonal to the principal stretch direction. The extensions at the corners
of the indenter increase the uniformity of the strain field over the indenter, resulting in a virtually
uniaxial stretch. Cells were seeded in the central 4 4-cm region of the membrane where strain was
uniform.
Stress fibers and Rho pathway
The GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation
of its effector proteins Rho kinase.
doi10.1073pnas.0506041102
Study on aortic endothelial cells.
Inhibitions of Rho, Rho-kinase.
Rho activity inhibited with C3 exoenzyme - C3 or by Y27632.
stress fiber formation was almost completely absent under unstretched condition (Fig.
A);
10% stretch at 1 Hz of these cells caused the formation of linear actin fiber bundles,
which were oriented parallel to the direction of stretch (Fig. B).
Control cells transfected with BSA (do not inhibit Rho activity).
Unstretched contained actin fibers not oriented in any particular direction (Fig. C).
Stretching resulted in the orientation of stress fibers perpendicular to the direction of
stretch (Fig. D).
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Stress fibers and Rho pathway
doi10.1073pnas.0506041102
Rho-kinase activity inhibited by Y27632 - Y27632.
Effect similar to inhibition with C3 exoenzyme
Stress fibers and Rho pathway
Immunostaining with an antibody specific for focal
adhesion kinase (FAK) revealed the association of
these fibers with focal adhesion-like structures in a
manner similar to that of stress fibers both in the
control cells transfected with BSA (Fig. 3C) and in cells
transfected with C3 (Fig. 3F).
doi10.1073pnas.0506041102
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Stress fibers and Rho pathway
Normally expressed Rho (GFP) vs overexpressed Rho (GFP+RhoV14)
Overexpression leads to orientation in lower stretch.
doi10.1073pnas.0506041102
Stress fibers and Rho pathway
Inhibitions of Rho, Rho-kinase, and mDia:
suppressed stress fiber formation, but
fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency).
In normal cells:
predominately perpendicular alignment of stress fibers to the stretch direction
the extent of perpendicular orientation of stress fibers depended on the
magnitude of stretch
In cells with Rho pathway inhibition:
stress fibers became oriented parallel to the stretch direction.
The activity of the Rho pathway plays a critical role in determining both the
direction and extent of stretch-induced stress fiber orientation in bovine
aortic endothelial cells.
The stretch-induced stress fiber orientation is a function of the interplay
between Rho pathway activity and the magnitude of stretching.
doi10.1073pnas.0506041102
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Shear stress and hydrostatic pressure – endothelial cells
Three different mechanical forces, shear stress, hydrostatic pressure
and cyclic stretch, acting on endothelial cells.
Biorheology, vol. 42, no. 6, pp. 421-441, 2005
Shear stress and hydrostatic pressure – endothelial cells
Dynamic change in actin filament structure of a single endothelial cells
exposed to fluid shear stress up to 300 min. (From Ohashi et al. [50] with
permission.)
Biorheology, vol. 42, no. 6, pp. 421-441, 2005
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Shear stress and hydrostatic pressure – endothelial cells
Typical fluorescent images of
actin filaments in bovine aortic
endothelial cells before and
after exposure to shear stress
of 2 Pa.
(a) Control,
(b) after 20 min,
(c) 40 min,
(d) 1 h,
(e) 3 h,
(f) 6 h.
(From Kataoka and Sato [29]
with permission.)
Biorheology, vol. 42, no. 6, pp. 421-441, 2005
Shear stress and hydrostatic pressure – endothelial cells
Fluorescence images of
actin filaments of EC
(a) control,
(b) hydrostatic pressure of
100 mmHg,
(c) shear stress of 2 Pa,
(d) hydrostatic pressure of
100 mmHg and shear stress
of 3 Pa.
The direction of flow was
from left to right.
Biorheology, vol. 42, no. 6, pp. 421-441, 2005
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Bones
The secondary bones are discussed here:
Critical characteristics of the bone:
Mechanical properties:
Toughness - HOUŽEVNATOST (resistance to fracture)
Stiffness - TUHOST (resistance to elastic deformation - the material
returns to its original shape when the force is removed).
Strength - PEVNOST (resistance to plastic deformation - the material
does not return to its original shape when the force is removed).
Structural adaptation to changing external conditions.
Capacity of self-repair.
Bone – Capacity of self repair
Capacity of self-repair:
the combination of growth and remodeling (resorption and replacement of old
material),
Osteoclasts are permanently removing material.
Osteoblasts are depositing new tissue.
Continuous remodeling allows for structural adaptation to changing external
conditions, as well as the removal and replacement of damaged material.
DOI: 10.1146/annurev-matsci-070909-104427
Trends in BiotechnologyVol. 20 No. 8 (Suppl.),
2002
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Minerals provide hardness and fragility.
Proteins provide plasticity.
DOI: 10.1557/opl.2011.94
Soft and flexible/ductile.
Hard and fragile.
Bone
Aging-related changes such as excessive remodeling.
Consequent fractures can lead to significant mortality.
A primary factor is bone quality - characteristics of the bone matrix nano- and
microstructure that can influence mechanical properties.
Traditional thinking focused on bone quantity described by the bone-mass
or bone mineral density (BMD), defined as the amount of bone mineral
per unit cross-sectional area.
For example, the elevation in bone-remodeling activity, concurrent
with menopause in aging women, can lead to osteoporosis, a
condition of low bone mass associated with an increased risk of
fracture.
Reality – more complicated proces involving not only BMD but other
factors such as hierarchy.
DOI: 10.1146/annurev-matsci-070909-104427
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Bioinspired structural materials - Bone
Bone is composed of cells embedded in an extracellular matrix, which is an
ordered network assembled from two major nanophases (95% of the dry
weight of bone):
Collagen fibrils made from type-I collagen molecules (~300 nm long, ~1.5 nm in
diameter).
Calcium fosfate based hydroxyapatite (Ca10(PO4)6(OH)2)) nanocrystals (plateshaped,
50 nm × 25 nm in size, 1.5–4 nm thick) distributed along the collagen
fibrils.
The hydroxyapatite nanocrystals are preferentially oriented with their c
axis parallel to the collagen fibrils, and arranged in a periodic, staggered
array along the fibrils.
Hierarchical structure of bone.
The macroscale arrangements of bones are
either:
Compact/cortical (dense material found at the
surface of all bones).
Spongy/cancellous (foam-like material whose struts
are some 100 μm thick).
Compact bone is composed of osteons that
surround and protect blood vessels. Osteons
have a lamellar structure.
Each individual lamella is composed of fibers
arranged in geometrical patterns. These fibers
are the result of several collagen fibrils, each
linked by an organic phase to form fibril arrays.
Each array makes up a single collagen fiber.
The mineralized collagen fibrils are the basic
building blocks of bone.
They are composed of collagen protein molecules
(tropocollagen) formed from three chains of amino
acids.
DOI: 10.1146/annurev-matsci-070909-104427
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Bones – toughness and hierarchy
Submicrometer deformation mechanisms contribute intrinsically to the fracture
toughness of bone by forming plastic zones around crack-like defects, thereby
protecting the integrity of the entire structure by allowing for localized failure
through energy dissipation.
Micro- to macroscale dimensions, the toughness of cortical bone is associated with a
crack-tip shielding.
Toughening that arises during crack growth rather than during crack initiation.
Microstructure, especially the interfaces of the osteons, provide microstructurally
weak or preferred paths for cracking.
As these features have a specific alignment in bone, the osteons provide the basis for
the marked anisotropy of the fracture properties of bone (bone is easier to split and to
break) and for the fact that the toughness is actually lower in shear than in tension.
Bones – Toughness and collagen
In collagen fibrils the following mechanism
competes:
Molecular uncoiling - breaking of weak and strong
bonds between tropocollagen molecules,
Molecular stretching,
Intermolecular sliding,
Collagen is able to stretch up to 50% tensile
strain before breaking while reaching force
levels of more than 10 nN(permolecule) or 10–
20 Gpa stress.
DOI: 10.1146/annurev-matsci-070909-104427
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Bones - Toughness and collagen - Molecular Uncoiling
H-bond breakage at 10% to 20% strain provides one of the major
mechanisms that mediate the deformation of collagen fibrils:
It is a reversible process and may thus provide a means to dissipate energy
through large-deformation behavior of the soft-collagen bone matrix.
Aged collagen tends to show a high cross-link density, whereas young collagen
features few crosslinks.
The larger the cross-link density, the lower is the material’s ability to dissipate
energy without failure. At large cross-link densities, collagen fibrils tend to
involve molecular fracture and breaking of cross-links, leading to increasingly
brittle material behavior.
DOI: 10.1146/annurev-matsci-070909-104427
Bones - toughness and collagen – Fibrillar sliding of mineralized
collagen fibrils
Importance of a mineralization of collagen:
Figure shows a tensile test of a tropocollagen molecule for deformation up to
40% strain.
Mineral phase has an elastic modulus that is more than an order of magnitude
higher than that of collagen, the presence of the hydroxyapatite phase is critical
to the stiffness of bone.
Slip at the hydroxyapatite/tropocollagen interface initiate the glide
between tropocollagen molecules and between hydroxyapatite particles
and tropocollagen molecules.
This glide enables a large regime of dissipative deformation once yielding
begins, thus effectively increasing the resistance to fracture.
DOI: 10.1146/annurev-matsci-070909-104427
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Bones - toughness and collagen – Fibrillar sliding of mineralized
collagen fibrils
Young’s modulus, tensile yield strain, and fracture strength for mineralized
collagen fibrils are 6.2 GPa, 6.7%, and 0.6 GPa, respectively, as compared
with the corresponding values of 4.6 GPa, 5%, and 0.3 GPa, respectively,
for pure-collagen fibrils.
DOI: 10.1146/annurev-matsci-070909-104427
Bones - toughness and collagen – Fibrillar sliding of collagen fiber
arrays
The long (>5–10-μm) and thin (∼100-nm) mineralized collagen fibrils are
twisted into collagen fibers, which are “glued” together by a thin layer (1–2
nm thick) of extrafibrillar matrix.
When the tissue is externally loaded in tension, the load is resolved into tensile
deformation of the mineralized fibrils and shearing deformation in the
extrafibrillar matrix.
DOI: 10.1146/annurev-matsci-070909-104427
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Bones - toughness and collagen – Fibrillar sliding of collagen fiber
arrays
Extrafibrillar matrix:
Composed of noncollagenous proteins, such as osteopontin, and proteoglycans,
such as decorin.
Properties similar to those of a glue layer between the fibrils—it is relatively
weak but ductile and deforms by the successive breaking of a series of
sacrificial bonds.
Specifically, the separation of individual fibrils and the larger fibers during
deformation and fracture is resisted by this macromolecular glue via sacrificial
bonds that break at a fraction (∼0.1–0.5) of the force required to break the
backbone of the macromolecules.
The matrix may also be partially calcified, which would increase its shear
stiffness and reduce its deformability.
DOI: 10.1146/annurev-matsci-070909-104427
Bones - Toughness – Microcracking
At several length scales from the submicrometer scale to a scale of tens of
micrometers, the proces of microcracking in bone provides the prevalent
mechanism of microscale deformation.
Microcracking effects:
Microcracking is a process of plastic deformation.
Essential phenomenon for the development of the most potent extrinsic
toughening mechanisms, notably crack bridging and crack deflection, that
predominate at larger length scales.
Crucial role in signaling the remodeling of the bone, which occurs in so-called
basic multicellular units (BMUs), i.e., combination of cells that are able to
remove (osteoclasts) and form (osteoblasts) bone tissue.
DOI: 10.1146/annurev-matsci-070909-104427
DOI: 10.1093/ageing/afl081
doi:10.1016/j.bone.2005.01.023
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Bones - Toughness – Constrained Microcracking
In cortical bone, the path of least microstructural resistance is invariably
along the cement lines which are the hypermineralized interfaces between
the bone matrix and secondary osteon structures.
These regions are therefore preferential sites for major microcracks to form,
particularly as bone ages and the osteon density increases with remodeling.
These microcracks thus have a typical spacing in the tens to hundreds of
micrometers and are aligned primarily along the long axis of the bone, an
orientation that directly results in the strong anisotropy of toughness in bone.
Fracture of hydroxyapatite crystals surrounding collagen fibers or
delamination at the crystal/fiber interfaces has been suggested as the
cause of such microcracking damage.
Importance of microcracking extrinsically is that it results in both crack bridging
and crack deflection, which are the most potent toughening mechanisms in
bone.
DOI: 10.1146/annurev-matsci-070909-104427
Bones - Toughness – Crack deflection, twist and bridging
DOI: 10.1146/annurev-matsci-070909-104427
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Bone tissue engineering – Critical bone defects
Critical bone defects need intervention therapy to achieve recovery.
Critical bone defects is regarded as one that would not heal spontaneously
despite surgical stabilization and requires further surgical intervention,
such as autologous bone grafting.
General guidelines that have been suggested in the literature include defect
length greater than 1–2 cm and greater than 50% loss of the circumference of
the bone .
However, this is impacted upon by the anatomic location of the defect and
the state of the soft tissues surrounding it.
Segmental defects of the femur often have a good soft-tissue
environment and spontaneous healing of segmental defects 6–15-cm
long has been reported.
By contrast, poor outcomes with the lack of spontaneous healing
have been reported with much smaller defects in the tibia, when the
defect size is greater than 1–2 cm and greater than 50% of the
cortical circumference.
doi.org/10.1016/j.actbio.2018.08.026  doi: 10.1097/BOT.0000000000000978
Bone tissue engineering – Critical bone defects
Interventions:
‘‘gold standard” for intervention - graft materials - limited by supply.
Fabrication methods with limited control of the pore shape, architecture,
porosity, or interconnectivity.
Extrusion-based processes - fabrication accuracy on tens and
hundreds of micrometers
Freeze drying
Fiber bonding
Particulate/salt leaching
Emulsification
Phase separation/inversion
doi.org/10.1016/j.actbio.2018.08.026  doi: 10.1097/BOT.0000000000000978
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Materials for bone substitutes
For metal bone substitutes:
• selective laser melting (SLM),
selective laser sintering (SLS),
• sintering,
• perforating titanium sheet,
• capsule-free hot isostatic
pressing (CF-HIP).
Polymer and ceramic
• Porogen leaching,
• freeze drying,
• 3D printing of successive
fibre/strut layers,
• electrospinning,
• gas foaming.
DOI: 10.1039/c7tb00741h
Markers of bone regeneration
During bone regeneration, cells proliferate and differentiate into osteoblasts which deposit a
collagen matrix that becomes mineralized.
Proliferation, takes place in the first days after seeding and consists mainly of cell division. Cells
are able to migrate.
After proliferation, cells start to differentiate into osteoprogenitor cells until the end of the
second week, and the release of alkaline phosphatase (ALP) increases.
In two weeks after the differentiation stage, osteocalcin (OCN) and osteopontin (OPN) are
produced and secreted by the cells, indicating the presence of osteoblasts.
When the collagen matrix is synthesized by osteoblasts, biomineralization is initiated and
mineral crystals are formed within the collagen matrix.
In parallel with the proliferation and differentiation of cells, blood vessels form from existing
vessels (angiogenesis). These vessels create a vascular network to provide oxygen and nutrients
to the cells and developing tissue within the bone substitute. This network provides stem cells
needed for bone regeneration and direct the differentiation of endothelial cells and
preosteoblasts.
DOI: 10.1039/c7tb00741h
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Optimization of scaffold structures
Porosity
Supports cell migration into the scaffold and improves the available surface
area for cell–scaffold binding and interaction with the surrounding tissues.
Conflicting issues that high porosity (30-50%) cannot simultaneously attain cell
migration and growth whilst maintaining optimal mechanical strength.
Compromise must be made among strength, bone integration, and porosity.
doi.org/10.1016/j.actbio.2018.11.039
https://biomaterials.ca/#!/abstracts/view/114242
Polycaprolactone (PCL) and borophosphosilicate (B2O3-P2O5SiO2)
glass (BPSG)
Compared to polycaprolactone, cells on the hybrid material
displayed enhanced spreading, focal adhesion formation, and
cell number, consistent with excellent biocompatibility.
BPSG
Optimization of scaffold structures
Pore size
The minimum pore size for a scaffold is approximately 100 nm because of cell
size, transport, and migration requirements.
Typically between 100 nm and 300 nm to allow cell penetration, migration, and
growth, as well as an optimal tissue vascularization
Smaller pores leads to low seeding efficiency and inhomogeneity.
Higher pores leads to cells tend to escape from the structure.
However, there is not yet a clear consensus on the effect of optimal pore size
on optimized mechanical and osteogenic performance.
doi.org/10.1016/j.actbio.2018.11.039 https://doi.org/10.1016/j.actbio.2013.12.042
The collagen porous scaffolds
prepared with ice particulates in the
range of 150–250 lm showed the
highest promoting effect on the
gene expression and the production
of
cartilaginous matrix proteins as well
as on cartilage regeneration.
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Pore size and Seeding effciency
The seeding efficiency depends on:
The number of attachment sites within a porous biomaterial:
Small pore size:
Cells aggregate at the seeding surface.
With an increased pore size:
The surface area within the structure decreases – thus there is less
attachment sites for the seeded cells.
The permeability of the porous biomaterial increases - this is
associated with a higher flow rate, which reduces the time for cell
attachment to the surface of the structure during seeding.
The available time for cells to attach to the surface.
DOI: 10.1039/c7tb00741h
Pore size and Cell viability, Proliferation and Migration
Pore size smaller than 100 nm:
Cells are more likely to aggregate and block the way for oxygen and nutrients to
the centre of the scaffolds.
Pores smaller than 100 nm should be avoided to prevent cell death.
Restricted cell migration was observed in porous biomaterials with small
pores, while cells can migrate more easily and distribute homogeneously
when a structure contains bigger pores up to 500 um.
DOI: 10.1039/c7tb00741h
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Pore size and Cell differentiation, Vascularization and Mineralization
Osteogenic differentiation occurred more in large pores.
The cells tend to be more spread in large pores compared to small pores. This
morphology is thought to promote osteogenic differentiation
Pores larger than 400 µm are preferable for blood vessel formation and
consequently for the delivery of oxygen and nutrients to the cells inside
the bone substitute.
Porous biomaterials with larger pores were found to have a better and
higher distribution of calcium and mineral deposition parallel to the pore
walls in vitro.
This could be an effect of the alignment of cells with the pore walls, higher cell
viability, distribution, and proliferation rate in structures with large pores.
DOI: 10.1039/c7tb00741hDOI: 10.1039/c7tb00741h
Pore shape
The geometry of pores within a bone substitute can be, among others,
spherical, rectangular, square, hexagonal or trabecularlike.
(a) Titanium; (b) Starch poly(ε-caprolactone); (c) Poly(lactide-co-glycolide); (d) bioactive
glass; (e) Poly(propylene fumarate); (f) collagen-apatite; (g) Mesoporous bioactive
glass; (h) Silk Fibroin
DOI: 10.1039/c7tb00741h
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The pore size and shape affect the mechanical properties of porous
biomaterials, as they determine the dimensions and orientation of the
struts or fibres and, thus, the stress distribution inside those structural
elements.
Scaffolds with a ladder-like structure and rectangular pores and scaffolds with
large spherical pores collapse more easily than porous biomaterials with
smaller uniform round pores.
DOI: 10.1039/c7tb00741h
Bone tissue engineering - Macrostructure
Macrostructure influence :
Mechanical property.
Degradation – connected to surface area and
porosity.
e.g. hollow-struts-packed possess a
faster degradation rate owing to their
large surface area and high porosity.
Permeability - water-uptake ability and water
movement.
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Bone tissue engineering - Macrostructure
Macropore geometry:
Cells prefer a radius of curvature much larger than that of the cells themselves.
Murine osteoblast-like cells exhibited curvature driven migration,
proliferation, and differentiation, thereby resulting in initial tissue
formation occurring at the corners.
High curvature leads to mechanical forces in cells, proved by the formation
of actin stress fibers at the tissue–fluid interface, which enhance further
tissue growth.
doi.org/10.1016/j.actbio.2018.08.026
Bone tissue engineering - Macrostructure - Multioriented hollow
channel structures
Multioriented hollow channel structures exhibited improved bone
regeneration:
Hollow channel structures led to a quick release of ions from the scaffolds.
Improved degradation offered more space for the formation of new bone
tissue.
Hollow tube provided a channel for oxygen and nutrition transport and cell
migration, thus benefiting the improvement in bone formation.
Bioactive ions released from the hollow-pipe-packed silicate bioceramic
scaffolds can also promote angiogenesis by inducing the migration of
endothelial cells.
Facilitate the infiltration of host blood vessels into the hollow channels and also
improve the delivery of stem cells and growth factors, thus contributing to
further tissue regeneration.
Promote osteoclast/immune cell invasion.
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Bone tissue engineering - Macrostructure - Multioriented hollow
channel structures
In vitro and in vivo results of the lotus root-like biomimetic scaffolds. (a, b)
SEM images of the attached BMSCs after culturing for 3 days.
(b) The yellow arrows show that the BMSCs adhered on the scaffolds
through numerous filopodia.
doi.org/10.1016/j.actbio.2018.08.026
Bone tissue engineering - Macrostructure - Multioriented hollow
channel structures
The effect of hollow channels
on stem cell delivery.
(a) SEM observations of
adherent rBMSCs on the
scaffold surface. Cells can be
observed in the hollow
channels of the BRT-H
scaffolds.
(b) The cells in the hollow
channels were still alive after
7 days of culture in vitro.
(c) Cell viability at day 1 and
day 7 of rBMSCs seeded in
the scaffolds.
(d) Scaffolds with luciferaselabeled
BMSCs were
transplanted into mice and
detected through in vivo
fluorescence imaging.
(e) Statistical results for the
relative fluorescence
intensity
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Bone tissue engineering – Microstructure  nanostructure
3D printing allow to prepare desirable macrostructure. The micro and nano
sized hierarchy must be subsequently be done by some of followed
methods:
Freeze-drying method
Hierarchically composite scaffolds were composed of ordered macropores of
the bioceramic scaffolds (first level: 1 mm) and micropores of silk networks
(second level: 50–100 lm),
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Bone tissue engineering – Microstructure  nanostructure
Self-assembly method
(a) Photos of 3D-printed pure
bioceramics (BC), DOPA-BC (2
mg/mL), DOPA-BC (4 mg/mL),
DOPA-BC (6 mg/mL)
scaffolds, respectively;
Optical photos of pure BC (b)
and 4 mg/mL DOPA-BC (c)
scaffolds on the top view;
SEM images of pure BC (d)
and 2 mg/mL DOPA-BC (e), 4
mg/mL DOPA-BC (f), 6 mg/mL
DOPA-BC (g) scaffolds,
showing a uniform nanolayer
composed of amorphous CaP
nanoparticles and
polydopamine on DOPA-BC
surface.
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Bone tissue engineering – Microstructure  nanostructure
doi.org/10.1016/j.actbio.2018.08.026
Spin coating method
TEM images for the coated BG (b) and MBG (e) layers on b-TCP scaffolds.
The MBG layer presents a well-ordered mesoporous structure with a
mesoporous pore size of 5 nm (e). Electron diffraction patterns reveal the
amorphous states of the coated BG (a) and MBG (d) layers as well as the
crystalline state of the substrate of b-TCP scaffolds (c, f)
Bone tissue engineering – Microstructure  nanostructure
Hydration process
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Bone tissue engineering – Microstructure  nanostructure
Combined a 3DP technique with a hydrothermal method to realize in situ
growth of molybdenum dissulfate MoS2 nanosheets on the strut surface of
bioceramic.
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3D printing for bone scaffolds
Fabrication through 3D printing
Stereolithography (SLA)
Selective laser sintering (SLS)
Fused deposition modeling (FDM)
Binder-based 3DP3DP
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3D printing for bone scaffolds
doi.org/10.1016/j.actbio.2018.11.039
4D printing for bone scaffolds
4D printing is a term referring to printed constructs that are designed to
change form and function after 3D printing based on response to an
environmental stimuli, thus offering additional capabilities and
performance-driven applications.
Using the same technologies as 3D printing but based on the development
of novel stimuli responsive biomaterials which interact with environmental
factors (e.g., humidity, temperature, or chemicals), and changes their
forms accordingly.
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Bone tissue engineering – bioceramic scaffolds
An ideal scaffold for bone tissue regeneration is designed to mimic the
structure and biological function of a healthy bone tissue in terms of both
chemical compositions and hierarchical structure as well as properties.
Bioceramic scaffolds (e.g., calcium phosphate ceramics, calcium silicate
ceramics, and bioactive glasses)
similarity to native bone inorganic components,
biocompatibility,
hydrophilicity,
bioactivity,
osteoconductivity, and osteoinductivity.
Bioceramic scaffolds have been designed with a hierarchical structure
consisting of the macro-, micro-, and nanostructures.
doi.org/10.1016/j.actbio.2018.08.026
Bone tissue engineering – bioceramic scaffolds
Calcium phosphate bioceramic scaffolds:
Certain calcium phosphate ceramics are osteoinductive in the absence of
supplements.
Unadeqate mechanical property limits the clinical application.
Calcium silicate bioceramic scaffolds CS (CaSiO3)
Bioactive glasses
Hydroxyapatite (HA) (Ca10(PO4)6(OH)2)
Chemical composition same as that of the main bone components
Leading to positive influences on adhesion and proliferation of
osteoblasts.
Slow degradation rate and poor mechanical strength as well as
fracture toughness of pure HA hinder complete bone formation and
possibly increases the risk of infection.
combined with different compositions like ZrO2, carbon fiber,
and Al2O3 to improve the mechanical characteristics. ZrO2, carbon
fiber, and Al2O3 are bioinert materials that reduce the bioactivity
of HA significantly.
combined with natural polymers – such as polylactide-coglycolide
acid (PLGA) – favorable biodegradability and good
biocompatibility.doi.org/10.1016/j.actbio.2018.08.026
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Bone tissue engineering – bioceramic scaffolds
Tricalcium phosphate (TCP),
First attempt to implant β-TCP as an artificial material to repair surgically
created fractures in rabbit bones was made in 1920 by the American
surgeon Fred Houdlette Albee.
Good biocompatibility and osteoconduction.
Ability to form a strong bone–calcium phosphate bond.
Better biodegradability than other biomaterial implants including HA.
Main mechanism of bioactivity of TCP is the partial dissolution and release
of Ca and phosphate ion products thus forming a biological apatite
precipitate on the surface of the bioceramic scaffold.
The bending strength and fracture toughness of β -TCP bioceramic
scaffolds are better than those of HA bioceramic scaffolds but still lower
than those of the human cortical bone.
Therefore, β-TCP bioceramic scaffolds cannot be used for loadbearing
implants.
Degradation rate of β-TCP bioceramic scaffolds cannot match the growth
rate of the new bone tissue.
doi.org/10.1016/j.actbio.2018.08.026
Bone tissue engineering – bioceramic scaffolds
Biphasic calcium phosphate (BCP)
Intimate mixture of hydroxyapatite and beta-tricalcium phosphate.
Good biocompatibility, bioactivity, and osteoconduction.
Combined with HA and β -TCP.
Compared with pure HA and pure β-TCP the BCP ceramics exhibit
controllable degradation rate, better biocompatibility, and enhanced bone
regeneration ability.
A large difference between HA and β -TCP scaffolds in sintering
temperature leads to poor sintering quality of BCP ceramics.
Due to low mechanical properties, BCP ceramics also cannot be used for
load-bearing bone tissue regeneration.
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Bone tissue engineering – bioceramic scaffolds
Doped elements
Zinc
The incorporation of Zn in HA ceramics improves the dissolution property.
Promotes osteoblast differentiation from bone marrow stromal cells in
vitro.
Ehances new bone formation in vivo by promoting osteoblast
mineralization.
Suppressing osteoclast differentiation by antagonism toward NF-jB
activation driven by TNFa, a suppressor of bone formation in vitro and in
vivo.
Strontium
pure-phase Sr5(PO4)2SiO4 bioactive ceramic scaffold prepared by combining
3DP technique with a solid-state reaction method.
Significantly stimulated the proliferation and angiogenesis-related
gene expression (KDR, VEGF, eNOS, and HIF1a) of HUVECs.
Could activate the Ca-sensing receptor, thus leading to the mitogenactivated
protein kinase signaling pathway and the activation of
inositol triphosphate production.
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Bone tissue engineering – bioceramic scaffolds
Manganese
Stimulation of cell adhesion and proliferation
Enhancement of the bioactivity of cartilage oligomeric matrix protein.
Promotion of osteogenic activity in vitro.
Especially for the regeneration of both cartilage and subchondral bone
tissues owing to the synergetic effect of Mn and Ca ions released from the
scaffolds, which can stimulate the maturation of chondrocytes and
regeneration of cartilage significantly.
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Bone tissue engineering – bioceramic scaffolds
Magnesium
Can affect vascularization and bone regeneration.
Scaffolds with modified core/shell printing setup by coaxial 3DP for
fabricating hollow-pipe-packed silicate bioceramic Ca7MgSi4O16
(BRT-H).
The released ions (including Mg, Ca, and Si ions) demonstrated
several effects for stimulating angiogenesis and osteogenesis.
In addition, the hollow structure of scaffolds showed a
synergistic effect with the released ions on promoting
vascularization
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Bone tissue engineering – bioceramic scaffolds
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