Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

CONTROLLED RELEASE

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 13

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011

Controlled drug delivery from scaffolds• Drug release upon matrix degradation• Drug release upon diffusion• Long-term maintenance of effective local

concentration• Localized effects ensured • Limited systemic effects

TÁMOP-4.1.2-08/1/A-2009-0011

Ideal scaffold• 3-dimensional and well defined microstructure• Interconnected pore network • Mechanical properties similar to those of

natural tissues • Biocompatible and bio-resorbable• Controllable degradation and resorption • Local sequestration and controlled delivery of

specific bioactive factors • Thus enhancing and guideing the regeneration

process

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ECM mimicry as a guide for scaffold design• ECM is the natural medium where cells

proliferate, differentiate and migrate• ECM is a highly organized dynamic

biomolecular environment where motifs governing cell behaviours are continuously generated and sequestered

• Motifs are locally released according to cellular stimuli

• Relase occurs on-demand upon degradation of the adhesion sites binding them to the ECM

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Growth factors and the ECM• Growth factors (GFs) are locally stored by

ECM• Storage in insoluble/latent forms • Specific binding with glycosaminoglycans

(e.g. heparins)• Elicit biological activity once released• ECM binding provides concentration gradient

important in morphogenesis

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Mimic the function of ECM• Future generations of TE scaffolds need to

have extended functionality and bioactivity • Synthetic bio-inspired ECM should broadcast

specific cellular events • The ability of controlled release of multiple

bioactive molecules will allow the control of cellular behaviour and successful regeneration

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Interspersed signals• Hydrogels (either natural or synthetic) have

been succesfully used for controlled release of bioactive protein compounds

• Molecules were simply mixed with the polymer and were entrapped upon gelation

• Natural (collagen, fibrin, hyaluronan) and synthetic (PEG-based, peptide-based) hydrogels have been used

• Release characteristic may modulated with crosslinking agents

• Solid-state scaffolds: fabrication method must be mild (to avoid protein denaturation)

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Immobilized signals• Modification of polymer scaffolds to interact with

signaling molecules: immobilization• Prolonged diffusion out of the scaffold platform• Reversible or irreversible binding to the polymer.• Released upon degradation of a linking tether or the

matrix itself• Determinants of the amount of bound signal and

release profile:– The number of binding sites– Affinity of the signal for sites– Degradation rate of the scaffold

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Signal delivery from cells• Inclusion of nucleic acids (NA) encoding the

desired protein• NA are introduced into target cells, which

then produce the desired proteins• Antisense oligos can be used to return

abnormal gene expression to a certain state• Synthetic polymers containing adhesion sites

(RGD) proved to be more effective in delivering the plasmid

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Protein delivery systems (DS) in TE• DS must prevent the protein from

inactivation or degradation• Fine-tuning of the release rate can be

achieved by modulating the composition, shape, and architecture of the platform

• Continous and pulsatile delivery• Biodegradable and non-degradable platforms

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Non-biodegradable systemsEthylene-vinyl acetate copolymers (EVAc) and

silicones:• Mass transport through polymer chains or pores is

the only rate-limiting step• Possible application in cell encapsulation preventing

them to interact with the immune system

Time

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• PLGA is a very versatile and widely used system• Poly-ortho esters are newly in the centre of interest

(no heating or solvents, injectable polymers)• Polyanhydrides usually undergo surface erosion

which has a favorable kinetics

Biodegradable systems

Time

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Controlled release profiles in biodegradable systems

Surface erosion

Bulk erosion

Correspondingrate

Typical releaseprofile

t t

dc(t)

/dt

Release rate

Amou

nt o

f dru

g re

leas

ed

t t t

dc(t)

/dt Toxic dose

c eff(t

)

Protein or smallmolecule drug

Protein or smallmolecule drug

Amou

nt o

f dru

g re

leas

ed Release rate

Correspondingrate

Typical releaseprofile

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On-off drug delivery systems• Pulsatile mode of protein and peptide release • Rapid and transient release of a certain amount of

drug molecules within a short time-period immediately after a pre-determined off-release interval

• Classified into “programmed” and “triggered” delivery systems (DS):– Programmed-DS: the release is governed by the

inner mechanism of the device – Triggered-DS: release is governed by changes in

the physiologic environment of the device or by external stimuli

• External stimuli involve temperature changes, electric or magnetic fields, ultrasounds or irradiation

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Programmed and triggered delivery systems• Synthetic polymers can be engineered to be applicable

in programmed delivery• Both surface and bulk-eroding systems may be used• Biggest interest in triggered delivery is the glucose-

sensitive insulin delivery• The “intelligent” system consists of immobilized

glucose oxidase in a pH-responsive polymeric hydrogel• In the gel, insulin is enclosed• Upon glucose diffusion into the hydrogel, glucose

oxidase converts it into gluconic acid• Lowering of the pH results in gel swelling and insulin

release

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Inclusion of drug molecules into scaffoldsPoly-methyl-methacrylate (PMMA) beads with antibiotics (mostly aminoglycosides):• Orthopedic and trauma surgery• Treatment of chronic osteomyelitis and/or ulcers• Bones and joints are „blind spots” of systemic

antibiotic therapy because the limited blood supply• PMMA beads release antibiotics gradually• High local antibiotic concentration can be achieved• Limited systemic side effects

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Inclusion of bioactive proteins into scaffoldsVEGF role in tissue vascularization:• Cells in hypoxic tissues secrete VEGF• Endothelial cells express VEGFR• Stimulates endothel proliferation• Directs endothelial cell migration• Tissue vascularization is critical in nutrition

and oxigenization of implanted TE constructs• Controlled VEGF delivery is in the focus of TE

research

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VEGF supports TE tissue vascularizationControlled VEGF delivery from alginate microparticles:• Bivalent cations mediate alginate crosslinking• VEGF encapsulation efficiency and delivery

ratio depends on the cation species (Ca2+ or Zn2+)

• Zn2+-crosslinked particles proved to be more toxic than Zn2+

• Mixture of Ca2+ and Zn2+ beads are the most favorable

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Support of tissue differentiation with bioactive proteinsBMP-2: • Key role in regulating osteoblast

differentiation• Recombinant hBMP-2 is dissolved in

aquaeous solution of polyethylene-oxide (PEO)

• rhBMP-2 solution is then added to scaffold material

• Scaffold materials include silk fibroin, PCLA, PEG, PLGA, collagen, etc.

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Experimental results with controlled drug delivery scaffolds – VEGF• Half-life of VEGF is 50 min, therefore

controlled release is critical• Controlled release is based on electrostatic

attractions between the carrier (acidic gelatine, IEP=5.0) and VEGF (IEP=8.6)

• Extent of gelatin cross-linking also influences release

• Up to 90% of total VEGF vas released within 30 days from sc. implants, 80% within the first 5 days.

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Clinical results with controlled drug delivery scaffolds – BMP-2• Use of BMP-2 filled collagen sponges in spinal

degenerative diseases to enhance post-operative bone fusion.

• BMP-2 treated patients regain the ability to self-care and mobility faster, their pain scores are significantly lower.

• Their mood and emotional control is also significantly better than that of control patients.

BIOSENSORS

Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 14

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011

Definition Biosensor is a device that transforms or detects a biological signal and transforms into a more easily detectable one.

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Concept of an implantable glucose sensor

Detector(potentially amobile phone)

Glucose sensorImplantable potentiostat

Type I

Signal

Type II

Insulin releaseGlucose sensor

Insulin container

Signal Signal

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Dexamethasone-loaded PLGA Microspheres

10m

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Model of biosensor-tissue interactions

Interphase

Microspherefor drug (TRM)

release

Tissue

Angiogenesis

Hydrogels + PEO

Endothelcell

Sensor

Biosensor

WBC

Angiogenic factor or other tissue response modifiers Fibrin CollagenSoluble proteins

RBC

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The “intelligent” system• Consists of immobilized glucose oxidase in a

pH-responsive polymeric hydrogel, enclosing a saturated insulin solution.

• As glucose diffuses into the hydrogel, glucose oxidase catalyzes its conversion to gluconic acid, thereby lowering the pH in the microenvironment of the membrane.

• Low pH causes swelling and insulin release.

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Development of reliable glucose biosensors require1. Novel electrodes are required to decrease

invasiveness of the implantable glucose biosensor2. Bioactive coatings are necessary to enhance the in

vivo life of the implantable glucose sensor3. Biosensor coating using electrospinning nanofibres

need to be developed4. Tissue responses are needed to be studied further

to optimize tissue responses to biosensor signals5. Angiogenesis around the glucose sensor need to be

increased to enhance detection potential of glucose levels and

6. Finally, novel biostable 3D porous collagen scaffolds need to be developed for tissue compatible biosensors