SUMMARY

Soft and injectable biomaterials are increasingly used in medical treatments, drug delivery and cell encapsulation for tissue engineering. These applications require rigorous investigations. In particular, the precise evaluation of mechanical properties is a key element for success because of their important role in the functionality of the biomaterial.

In tissue engineering, following the in vitro tissue growth and matrix biodegradation is highly important to ensure that the biodegradation rate coincides with new tissue regeneration at the defect site in vivo. Several techniques, such as rheometry, dynamic mechanical analysis (DMA), indentation testing and atomic force microscopy (AFM), can be used to evaluate the mechanical properties of hydrogels. However, these techniques do not allow non-destructive, contactless, sterile and long-term monitoring of mechanical properties, especially under cell culture conditions.

Rheolution Inc. has developed a new instrument, ElastoSensTM BIO2, which allows quantifying the viscoelastic properties of gels and soft materials in real time and for sustained periods of time without destroying the sample. The innovative technological principle behind this instrument enables contactless and non-destructive measurements.

ElastoSensTM BIO2 has strong potential in the field of biomaterials and tissue engineering because:

- Processes like gelation and degradation can be followed in real time under desired conditions and the samples can be exposed to suitable chemical and physical stimuli.

© Rheolution Inc. All rights reserved.

How to track long-term mechanical evolution of the same biomaterial sample?

gelation, hydrogels, mechanical testing, non-destructive & contactless testing,

ElastoSensTM BIO2: the first contactless instrument with detachable sample holder

Viscoelastic Monitoring of the Formation of Hydrogels by ElastoSensTM BIO2

For repeated non-destructive real-time testing of biomaterials and hydrogels

ThermalMeasures

BioChemistry

ElastoSensTM BIO2: real-time, non-destructive and contactless instrument for mechanical monitoring of gelation and degradation processes of hydrogels.

BiologicMaterials

TemporalMeasur.

SUMMARY

Soft and injectable biomaterials are increasingly used in medical treatments, drug delivery and cell encapsulation for tissue engineering. These applications require rigorous investigations. In particular, the precise evaluation of mechanical properties is a key element for success because of their important role in the functionality of the biomaterial.

In tissue engineering, following the in vitro tissue growth and matrix biodegradation is highly important to ensure that the biodegradation rate coincides with new tissue regeneration at the defect site in vivo. Several techniques, such as rheometry, dynamic mechanical analysis (DMA), indentation testing and atomic force microscopy (AFM), can be used to evaluate the mechanical properties of hydrogels. However, these techniques do not allow non-destructive, contactless, sterile and long-term monitoring of mechanical properties, especially under cell culture conditions.

Rheolution Inc. has developed a new instrument, ElastoSensTM BIO2, which allows quantifying the viscoelastic properties of gels and soft materials in real time and for sustained periods of time without destroying the sample. The innovative technological principle behind this instrument enables contactless and non-destructive measurements.

ElastoSensTM BIO2 has strong potential in the field of biomaterials and tissue engineering because:

- Processes like gelation and degradation can be followed in real time under desired conditions and the samples can be exposed to suitable chemical and physical stimuli.

- The same sample can be tested repeatedly over long periods of time to follow long-term changes in viscoelastic properties.

- The sample is kept completely sterile in the sample holder before, during and after the measurements. It can be disconnected from the instrument without the risk of contamination and stored in an incubator.

- A special sample holder can be connected to a bioreactor to expose the sample adequately and permanently to gas and nutrients for good cell survival and growth.

This study shows how ElastoSensTM BIO2 is used to measure the temporal evolution of the viscoelasticity of agar and chitosan hydrogels during the cross-linking process.

ElastoSensTM BIO2: PRINCIPLE & COMPARISON

ElastoSensTM BIO2 is a benchtop instrument that measures without contact, non-destructively and in real time the shear storage (G’) and loss (G’’) moduli of gels as function of time and temperature.

The patented technological principle behind this instrument is based on an original approach using propagative mechanical waves. The sample is contained into a cylindrical cuvette having a flexible bottom part. A small and gentle vibration of a few micrometers is applied to the sample and its response is measured using a laser probe without contact. Finally, the material response is processed to obtain the viscoelastic properties of the gel (i.e. shear storage and loss moduli). This process is repeated to characterize the mechanical evolution of the studied material.

The instrument allows connecting probes (such as pH) to perform simultaneous and complementary measurements on control samples.

GELATION OF AGAROSE AND CHITOSAN HYDROGELS

The first example shows the evolution of the elastic moduli over time during the gelation of two aqueous agar samples, measured by ElastoSensTM BIO2. The agar concentrations were 0.7 % (w/w) for the soft gel and 1.0 % (w/w) for the hard gel. The liquid samples were poured into the sample holder of ElastoSensTM BIO2 to initiate gelation, and the viscoelastic properties were characterized using the instrument. The sample volume was 2 mL, and the measurements were conducted at constant temperature of 22 °C every 10 s during 120 min. From Figure 3, we can clearly see that the gelation kinetics of agarose hydrogels (i.e. speed of hardening and maximum elasticity) depends strongly on the concentration of agar. The result shows that the cross-linking process is measured precisely by ElastoSensTM BIO2.

Fig. 3 Time evolution of the elastic moduli during the gelation of two agarose hydrogels at different concentrations, measured by ElastoSensTM BIO2

© Rheolution inc. All rights reserved.

The second example shows the evolution of the viscoelasticity of chitosan-based hydrogel during the gelation process. An aqueous solution of chitosan (Marinard Biotech, Rivière-au-Renard, QC, Canada) was mixed with a gelling agent to induce gelation of the polymer (final concentration 2 % w/w). After rapid mixing, the solution (2 mL) was poured into the ElastoSensTM BIO2 sample holder, which was then placed in the thermal chamber of the instrument set to 37 °C. A thin film of distilled water was added to cover the sample and to avoid drying. Figure 4 shows the evolution of storage modulus (G’) and loss tangent (tan(δ)=G’’/G’) of a chitosan sample as function of time over 60 minutes. The results show an increase in the elastic modulus of the hydrogel and decrease in the loss tangent accompanying a gradual shift from viscous liquid to soft viscoelastic solid.

APPLICATIONS OF ELASTOSENSTM BIO2

ElastoSensTM BIO2 is a suitable measurement tool to characterize short and long term viscoelastic evolution of hydrogels and synthetic soft biomaterials. In the context of hydrogels used for regenerative medicine and tissue engineering, the main advantages of this instrument are the following:

Contactless measurementsThanks to its new vibrational technique, ElastoSensTM BIO2 characterizes samples without contact using an optical probe. This allows keeping the sample sterile during the measurement.

Non-destructive measurementsThe instrument applies gentle micrometric vibrations to the sample to prevent any damage or structural changes.

Detachable sample holder The sample is contained in a sample holder that can be detached from the instrument. This allows performing measurements on the same sample over long periods of time for observing long-term viscoelastic changes.

A bioreactor compatible sample holderThe sample holder of ElastoSensTM BIO2 has been designed to be connected to a bioreactor in order to study the mechanical evolution of 3D cell cultures in hydrogels for tissue engineering.

ElastoSensTM BIO2, an instrument simple to use with unique and unmatched capabilities, is particularly adapted to biologists, chemists and material scientists involved in the development and production of hydrogels for tissue repair and medical use, in the development of engineered tissues for regenerative medicine, and in the study of cell growth in gel matrices. All these fields can benefit from fast measurements which can be repeated on the same sample for long-term testing of materials, and the samples can be switched easily to maximize the instrument use. The tests are simple and programmable and the interpretation of results is intuitive. Non-destructive testing and the use of detachable sample holders accelerate studies and increase the statistical significance of results by reducing the variability in the mechanical measurements on biological materials.

All these capabilities are made possible by the new technology of ElastoSensTM BIO2. An instrument designed to overcome challenges in the development of biomaterials and their medical implementation.

Fig. 4 Time evolution of the viscoelastic properties of a chitosan hydrogel during cross-linking measured by ElastoSensTM BIO2

ElastoSensTM BIO2 delivers viscoelastic information that is identical to classical rheometry (shear storage and loss moduli), but measured without contact. Figure 2 shows a comparison of the results of the gelation of agarose (Noble Agar, Sigma, A5431, 1.4% (w/w)) measured by ElastoSensTM BIO2 and a rotational rheometer.

Fig. 2 Comparison of ElastoSensTM BIO2 with rotational rheometry.

Time (min)0 10 20 30 40 50

Shea

r mod

ulus

, G' (

kPa)

02468

1012141618

ElastoSensTM Bio2

Anton Paar MCR 301

G’

Time (min)0 10 20 30 40 50

Phas

e an

gle,

Tan

(δ)=

G''/

G'

0.01

0.1

1

ElastoSensTM Bio2

Anton Paar MCR 301

Tan(δ)

Fig. 1 Principle of ElastoSensTM BIO2: Non-contact and non destructive measurement of few micrometers amplitude vibrations

Time (minutes)

0 20 40 60 80 100 120

Shea

r ela

s�c

mod

ulus

G (P

a)

0

5000

10000

15000

20000

25000

30000

Agarose - 0.7%Agarose - 1.0%

Time (min)

20 30 40 50 60

Shea

r ela

s�c

mod

ulus

G' (

Pa)

0

500

1000

1500

2000

2500

Tan(δ )

0.0

0.2

0.4

0.6

0.8

1.0

G’

Tan(δ)

The second example shows the evolution of the viscoelasticity of chitosan-based hydrogel during the gelation process. An aqueous solution of chitosan (Marinard Biotech, Rivière-au-Renard, QC, Canada) was mixed with a gelling agent to induce gelation of the polymer (final concentration 2 % w/w). After rapid mixing, the solution (2 mL) was poured into the ElastoSensTM BIO2 sample holder, which was then placed in the thermal chamber of the instrument set to 37 °C. A thin film of distilled water was added to cover the sample and to avoid drying. Figure 4 shows the evolution of storage modulus (G’) and loss tangent (tan(δ)=G’’/G’) of a chitosan sample as function of time over 60 minutes. The results show an increase in the elastic modulus of the hydrogel and decrease in the loss tangent accompanying a gradual shift from viscous liquid to soft viscoelastic solid.

APPLICATIONS OF ELASTOSENSTM BIO2

ElastoSensTM BIO2 is a suitable measurement tool to characterize short and long term viscoelastic evolution of hydrogels and synthetic soft biomaterials. In the context of hydrogels used for regenerative medicine and tissue engineering, the main advantages of this instrument are the following:

Contactless measurementsThanks to its new vibrational technique, ElastoSensTM BIO2 characterizes samples without contact using an optical probe. This allows keeping the sample sterile during the measurement.

Non-destructive measurementsThe instrument applies gentle micrometric vibrations to the sample to prevent any damage or structural changes.

Detachable sample holder The sample is contained in a sample holder that can be detached from the instrument. This allows performing measurements on the same sample over long periods of time for observing long-term viscoelastic changes.

A bioreactor compatible sample holderThe sample holder of ElastoSensTM BIO2 has been designed to be connected to a bioreactor in order to study the mechanical evolution of 3D cell cultures in hydrogels for tissue engineering.

ElastoSensTM BIO2, an instrument simple to use with unique and unmatched capabilities, is particularly adapted to biologists, chemists and material scientists involved in the development and production of hydrogels for tissue repair and medical use, in the development of engineered tissues for regenerative medicine, and in the study of cell growth in gel matrices. All these fields can benefit from fast measurements which can be repeated on the same sample for long-term testing of materials, and the samples can be switched easily to maximize the instrument use. The tests are simple and programmable and the interpretation of results is intuitive. Non-destructive testing and the use of detachable sample holders accelerate studies and increase the statistical significance of results by reducing the variability in the mechanical measurements on biological materials.

All these capabilities are made possible by the new technology of ElastoSensTM BIO2. An instrument designed to overcome challenges in the development of biomaterials and their medical implementation.

Heating speed and stability

Temperature range

Mechanical & Physical

Temperature

Shear Elastic Modulus

Precision

0 Pa to 100 MPa

0.1 Pa

2°C to 65°C

Precision 0.5°C

Spec. of ElastoSensTM BIO2

Temporal

Temporal resolution 1 second to 120 minutes

10 seconds to 1200 hoursMeasurements duration

ElastoView

Interface Simple interface with touchscreen

Test settings, post-processing, real time results display, archiving and data transfer

Software

Simultaneous 3 samples (optional)

Swelling measurement YES

Programmable ramps YES

ACKNOWLEDGMENT

This study has been performed in collaboration with Pr. S. Lerouge and Dr. C. Ceccaldi from École de technologie supé-rieure (ÈTS, Montreal) and with the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC Engage grant).

Rheolution Inc.5333 Casgrain Ave., Suite #712 Montreal, QC, H2T 1X3, Canada

+1 514-586-2006 www.rheolution.com informations@rheolution.com