Collagen scaffold production: The freeze drying method

Published: 24-Sep-2018

Collagen bio-scaffolds are hugely beneficial to many medical fields, therefore it is essential that the process used to develop them is as robust and viable as possible. Here, Bio Pharma Group outline the main markets, applications and importance associated when freeze drying collagen bio-scaffolds and the care that must be taken during the freezing, as well as drying steps to maximise the efficiency and ice crystal structure/target porosity

Uses of collagen scaffolds in medicine

Collagen is the most abundant protein in the animal kingdom. It comprises 30% of the total protein found in the human body and is the key structural protein in the naturally occurring extracellular matrix (ECM) of various fibrous tissues including tendons, ligaments and skin; but also bones, corneas, blood vessels, cartilage and connective tissues. It is the glue that holds our bodies together, providing structural support and strength.

In vitro, collagen can be formed into highly organised, three-dimensional scaffolds that are biocompatible, biodegradable and non-toxic upon exogenous application. All of these features make collagen the material of choice for tissue engineering, regenerative medicine and wound healing. Every day, thousands of surgical procedures are performed to replace or repair tissue that has been damaged through disease or trauma.

Tissue engineering is a relatively new specialism that aims to regenerate damaged tissues by combining cells from the body with collagen scaffolds, which act as templates for new tissue growth. Collagen for medical products can be derived from a range of different sources – there is a growing influence from porcine, chicken and also jellyfish derivatives, but most commonly being that of bovine. It is used to guide and encourage tissue regeneration in sponge, thin sheet or gel form.

Collagen bio-scaffolds can be used in a wide variety of medical fields, but it’s not a case of one size fits all, so it’s essential to develop a process that makes them as robust and viable as possible for their end purpose.

They can be used:

  • In vivo (in a living organism) – to support the regrowth of tissue or bone after surgery or an injury.
  • In vitro (outside normal biological context) – for example, growing new organs from adult stem cells.

Some of the more typical medical applications which collagen scaffolds have been used for include:

  • Bone grafts – bio-scaffolds don’t compromise the structural integrity of bones and due to collagen’s triple helical structure, it is strong and cannot be broken down by enzymes. It is very important for the proper assembly of extracellular matrices.
  • Cosmetic surgery – collagen can be used to treat skin ageing by removing wrinkles and lines with dermal fillers, and can also help with stretch marks and some types of scars.
  • Wound dressing – as a significant component of skin tissue, collagen can benefit all stages of the wound healing process, including second-degree burns, by offering a feasible platform for the topical conveyance of cells into the wound bed.
  • Tissue regeneration and engineering – collagen has very good properties for tissue regeneration: controllable pore structure, permeability, hydrophilicity and stability in vivo.
  • Cardiac applications – collagen scaffolds can help to repair the heart after a heart attack.
  • Other areas include treatment of osteoarthritis and newer cancer therapies.

With freeze drying, it is possible to precisely manage how the ice crystals grow to control the shape and properties of the resulting collagen scaffold. This is achieved by controlling the freezing rather than drying elements of a cycle recipe. This has not always been fully understood, since over the years, emphasis has been on understanding the drying phases, where vacuum is introduced and managing efficient sublimation rate of the sample. Nonetheless, today's understanding confirms that the focal point of any recipe (where operators seek to manipulate the ice crystal structure and porosity of a particular sample) should be achieved during the initial freezing step.

It is widely acknowledged that fast (flash or quench) cooling produces smaller ice crystals with a poor ice crystal network, whereas slow cooling forms larger ice crystals and therefore larger pores. It is also essential to use a suitable cooling rate and container with an appropriate heat transfer coefficient to form the desired pore size. An additional annealing step, (where the temperature of the sample is raised after initial freezing) could be added to increase ice crystal size. Or to even out the distribution of ice crystal size and make the material more homogenous.

Choosing the right kit

It is therefore imperative that appropriate attention is given when selecting a machine capable of replicating the shelf temperature control needed to enable multi-step freezing to take place in-situ of the machine. Freeze dryers not capable of multi-step freezing and annealing may be unable to follow the specific/optimum recipe designed for a product, which might then impact product integrity and efficacy. Increasingly, companies generally prefer systems with the functionality to control all stages of the cycle.


Cross-linking is another important point of consideration when reviewing collagen based processing as this is a bond that links one polymer chain to another. Precaution should be taken when cross-linking is applied, because this could potentially generate a micro-collapse. Typically, this could occur in formulations that contain a mixture of crystallising and non-crystallising (amorphous) components, but specifically for collagen-based products, this may occur when they are impregnated with other solutes, then lyophilised.

The most common cross-linking agents are:

  • Acetic acid – Freeze drying can be a good means of removing any acetic acid remaining due to its volatility (Vapour pressure is 11.6 Torr at 20°C)
  • EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) in the presence of NHS (N-hydroxysuccinimide) – forms “zero length” crosslinks (form protein conjugates without adding additional spacer arm atoms between two conjugated molecules)
  • Glutaraldehyde (GTA) – takes the form of long polymer chains

It is important to choose the correct cross-linking agent for your specific purposes and be aware that some amount of the cross-linking agent will likely remain in the final freeze dried product.


Freeze drying provides the opportunity to produce highly aligned scaffolds. Matrix and cellular alignment are critical factors in the native function of many tissues, so it’s desirable to reproduce this in scaffolds for tissue engineering.

Structural alignment gives mechanical strength to load-bearing tissues such as cardiac muscle or the muscle lining of blood vessels, and it also provides a guidance field for migrating cells or processes during wound healing and tissue regeneration. Alignment can be easily achieved by freezing and lyophilising collagen, which has been assembled to form a fibrillar hydrogel in a cylindrical conduit with high aspect ratio, negating the need for additional specialised equipment, extensive incubation or denaturing of the collagen which may be required for other methods of scaffold fabrication. For these reasons, freeze drying has, and continues to be, a highly popular technique for companies and operators, alike.

Nonetheless, the functionality/capability of the freeze dryer used shouldn’t be overlooked either, because the importance of controlling the freezing rate to assist in the development of the operators desired ice crystal size and network is inherent.

Many systems available are only to control the drying phases of the process, but product must be loaded pre-frozen. However, some dryers, such as the Cuddon Freeze Dry series of instruments are able to offer freeze in-place, in situ of the unit, which has proved to be a popular function in this particular field.

As featured in International Labmate July 2018: Volume 43 Issue 5 – Pages 20 & 21

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