COLLECTION
& SELECTION

Collection of femoral heads from living donors harvested in France by orthopaedic surgeons. Clinical selection based on criteria recommended by the Biomedicine Agency. Biological selection based on regulatory serological selection.

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01

TRANSPORTATION

Conveyance of the femoral heads and blood tubes to BIOBank is done by a specialized transporter with a -30°C transport system. The cold chain is monitored by an electronic probe temperature recording device.

02

Supercrit®

The BIOBank Supercrit® process is based on the delipidation of bone tissue by a non-toxic fluid, CO2 in supercritical state, combined with a chemical oxidation of the residual proteins contained in the pores of the cancellous tissue.

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03

DISTRIBUTION & STORAGE

BIOBank bone grafts are delivered directly by our company on medical prescription or through your local distributor. They are protected by a double sealed sterile package and can be stored at standard room temperature, brightness and humidity conditions until their expiry date, while the package remains undamaged.

04

IMPLANTATION

An implantation with a BIOBank graft meets strict traceability and usage rules in terms of patient information and graft use.

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05

The treatment of bone tissue

Cryopreserved bone allografts have been widely used and are still used in orthopaedic surgery for the treatment of significant bone defects, specifically in hip and knee arthroplasty revisions. The broadening of their indication to small bone fillings has favoured the development of techniques that aim to offer adapted shapes. Oral bone surgery with implant placement is, in this regard, a boom sector in the use of bone allografts.

Various treatment methods have been developed since the end of the 1980s to devitalise bone tissue and align it with the three required basic properties: active viral inactivation (no longer only just through donor selection), storage at ambient temperature made possible by a dehydrated state and osteoconduction improved by the cleaning of the bone trabeculae.

In the usual procedures, a strong organic solvent (chloroform or acetone) is used to degrease the cancellous bone tissue which contains many adipocytes in its medullar cavities. This lipid fraction actually represents nearly 60% in mass of a femoral head after elimination of the cartilage. Its elimination is made even more difficult with the density and thickness of the trabecular network (Figure 1).

Figure 1: Application of the Supercrit® process on a whole femoral head: illustration of the capacity to obtain both effective cleaning and preservation of the initial bone volume.

Furthermore, the presence of a large quantity of lipids entails poor wettability of the tissue, preventing or interfering with the action of aqueous oxidants later introduced for the viro-inactivation. Their spread in the bone tissue is limited, which reduces their ability to guarantee the virus inactivation for large-size bone fragments, such as whole femoral heads.

Besides, the use of these degreasing solvents requires a phase of elimination of the dissolved grease. It is carried out especially through aggressive physical treatments such as high pressure jet of water or centrifugation. These organic solvents can also leave undesirable toxic residue if not properly eliminated.

Finally, another disadvantage of using organic solvents is their relative low efficacy. Lipid residues after usual treatment still represent a none negligible fraction of the mass of the final product. This results in an imperfect wettability that inhibits the osteoconduction, hindering the vascularisation of the graft in situ and lets cytotoxic residue to appear during gamma irradiation.

We should consider that elimination of grease by deep cleaning of the bone’s trabecular network is the cornerstone of all the treatment later stages and an essential prerequisite for the osteoconduction expected by the surgeon.

A ground breaking technology with supercritical carbon dioxide has therefore been developed to be applied to human bone tissue, under the name Supercrit® process.

Principle and implementation of the Supercrit® process

Usual bone allograft processing uses strong organic solvents for the cleaning. BIOBank is the first tissue bank who has developed and used supercritical CO2 as a high-performance, non-toxic extraction fluid. The Supercrit® process is the combination of a degreasing step by supercritical CO2 and a gentle chemical oxidation of the residual proteins. This procedure has unmatched performances in terms of safety and preservation of the bone matrix.

In its principle, beyond a critical pressure and temperature, CO2 is no longer in gaseous or liquid state, it is in another state named supercritical fluid (Figure 2). As a liquid, it has a high density and retains a high solvating power. Another useful property is its low viscosity, which guarantees a high diffusivity (9).

Figure 2: Diagram of phase changes of carbon dioxide. Beyond 31.1°C and 73.8 bars, CO2 is in a state known as supercritical.

These two properties, high diffusivity and solvating power, make this fluid very useful for the extraction of apolar compounds contained in a porous matrix such as cancellous bone tissue. Furthermore, its supercritical coordinates are mild (critical pressure of 7.38 MPa; critical temperature of 31°C) and do not entail any denaturation of proteins like bone collagen.

Non-polar molecules as hydrogen carbides, oils and more generally all lipids are soluble in CO2 in its supercritical state. Conversely, polar molecules, amino acids and proteins are poorly soluble. This last property is essential because it preserves the integrity of the collagen contained in the bone tissue, in which it accounts for approximately 30% of the weight.

Thanks to the efficacy of the degreasing, the wettability of the bone tissue is completely changed. It facilitates the penetration of aqueous chemical products, which are then used for the viro-inactivation. It reduces the time necessary for them to act and contributes to a better preservation of the bone tissue’s mechanical properties.

The performances of the Supercrit® process in terms of reduction of a pre-inoculated viral load have been validated (5, 6). The virus inactivating power of the Supercrit® process far exceeds the security assurance level (SAL) of 10-6 required to guarantee the sterility of bone grafts (Figure 3). The evaluation of the elasticity and compressive strength of BIOBank bone grafts, performed according to several protocols, has demonstrated the specificity of the Supercrit® process to better preserve bone tissue architecture and density than others (1, 2, 3).

Figure 3: Capacity of the Supercrit® process to reduce a pre-inoculated viral load carried out on the 5 main virus families (source: Pasteur Texcell institute).

At the end of the Supercrit® procedure, the trabecular bone tissue is perfectly clean and retains its initial architecture and volume (Figure 4).

Figure 4: Illustration of the efficacy of the BIOBank procedure on a femoral head before, during, and after the implementation of the Supercrit® process.

All tissue handlings are done in a laboratory specially designed for the aseptic treatment of tissues. The work environment consists of clean rooms with a controlled atmosphere, equipped with specific high-tech materials. A trained and qualified staff implement all stages of the Supercrit® process.

After cleaning, drying and packaging, the sterile state is obtained by irradiation with gamma rays at a sterilising dose of 25 to 30 kGy on grafts protected by a double primary packaging. As for implantable medical devices, the sterilisation by gamma irradiation is performed by an accredited service provider, and validated in accordance with standards EN 552 and ISO 11737-2. Apart from its sterilising efficacy and its strong penetrating power, this method is traditionally used for bone allografts as it doesn’t cause the material to heat up and doesn’t generate any toxic residue.

Mode of use and mechanism of action of BIOBank bone grafts

The rehydration is an important step, necessary for the bone graft to recover its initial elasticity. It can be done with sterile physiological serum or, even better, with the patient’s blood. Indeed, its brings blood cells and proteins that, on the formation of the blood clot, will constitute a network favourable to cell adhesion and activation of factors involved in the tissue healing process. The rehydration with blood also enable a cohesion of the powder grains, which facilitates handling.

Like any inert biomaterials without growth factors, the BIOBank bone allograft acts by osteoconduction. The grafted bone matrix is invaded by a neo-vascularisation from the recipient bone bed, which will provide the bone cells needed to transform the bone graft into a living and functional bone tissue (7, 8).

The difference of BIOBank allograft from other osteoconductor materials lies in the respect of the bone tissue composition, the preservation of its architecture (Figure 5) and mechanical properties, and in the particularly high wettability resulting from the efficacy of the Supercrit® process.

Figure 5: Cross-section of a cortico-cancellous bone block with a scanning electron microscope (x60).

The patient’s bone cells will thus penetrate a familiar environment from a biochemical point of view and will be able to spread and drop off an osteoid tissue on the surface of the bone trabeculae, without inhibition or delay. Thenceforth, the mechanism known as resorption/apposition starts within the first hours after the grafting. The absence of cell debris facilitates this spread and considerably limits the cell and humoral foreign body reaction. The kinetic of osteoconduction is faster than with materials composed of a single mineral phase or whose cleaning and wettability are less efficient.

These properties that combine efficacy and tolerance are confirmed by histological analyses regularly performed during implant placement (Figure 6).

Normally, 4 to 6 months after the grafting of a cancellous bone powder in a sinus or a post-extraction socket, a picture of the bone remodelling process includes the following elements:

  • A close juxtaposition between the residual fragments of the graft and a living neoformed bone, with a balance between the two tissues, the graft particles acting as bridges within the new bone tissue
  • A cellularized bone marrow without giant or macrophagic cells
  • Presence of blood vessels involved in the tissue metabolism
  • Osteoblast cells fronts on the surface of the osteoid tissue undergoing mineralisation
  • Some rare osteoclasts may subsist, testifying to a certain stability or even the end of the active resorption phase

Figure 6: Histological analysis of a sinus biopsy by Dr Carole Leconte (Paris).
Hematoxylin-Eosin-Saffron staining performed by Novotec (Lyon) - *: graft particle, ☆: neoformed bone, -> osteoid tissue, bm: bone marrow, ob: osteoblast, oc: osteoclast.

The graft’s resorption process is not completed, which isn’t a problem because the residual particles behave as living bone in biomechanical terms. The notable point is the obtaining of real cohesion between the two bones types within the grafted volume. The graft’s particles have been really assimilated in a trabecular network of functional, neoformed bone.

Conclusion

All of the in vitro and pre-clinical studies validate the pertinence of the Supercrit® process. Through its cleaning and viro-inactivation performances, along with the maintaining of the bone matrix, it is a modern and effective approach. The implementation of such a procedure is done within a complex organisation, requiring the mastery of a large number of skills and expertises while placing rigour at the centre of each stage.

The clinical experience available today demonstrates the interest in bone allografts and confirms that bone tissue is an exceptional natural biomaterial.

Bibliographical references

1Comparative ultrasound evaluation of human trabecular bone graft properties after treatment with different sterilization procedures.

VASTEL, L., MASSE, C., MESNIL, P., et al.

Journal of Biomedical Materials Research, 2009, Vol.90, N°1, p. 430-437.

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2Effects of gamma irradiation on mechanical properties of defatted trabecular bone allografts assessed by speed of sound measurement.

VASTEL, L., MASSE, C., MESNIL, P., et al.

Cell and Tissue Banking, 2007, vol.8, p. 205-210.

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3Effect of a supercritical C02 based treatment on mechanical properties of human cancellous bone.

MITTON, David., RAPPENEAU, Julien., BARDONNET, Raphaël.

European Journal of Orthopaedic Surgery & Traumatology, 2005, N°15, p. 264-269.

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4Effect of different sterilization processing methods on the mechanical properties of human cancellous bone allografts.

VASTEL, L., MEUNIER, A., SINEY,H., et al.

Biomaterials, 2004, N°25, p. 2105-2110.

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5Evaluation of the viral safety during manufacturing processe of human bone grafts. Evaluation carried out by using the whole femoral head.

Pasteur-Texcell

report number : 250/01/5375/01, 2003.

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6Viral inactivation of human bone tissue using supercritical fluid extraction.

FAGES, Jacques., JEAN, Eliane., FRAYSSINET, Patrick., et al.

Journal of Supercritical Fluids, 1998, N°13, p. 351-356.

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7Bone allograft and supercritical processing : effects on osteointegration and viral safety.

FAGES, Jacques., JEAN, Eliane., FRAYSSINET, Patrick., et al.

Journal of Supercritical Fluids, 1998, N°13, p. 351-356.

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8Histological integration of allogeneic cancellous bone tissue treated by supercritical CO2 implanted in sheep bones.

FRAYSSINET, Patrick., ROUQUET, Nicole., MATHON, Didier., et al.

Biomaterials, 1998, N°19, p. 2247-2253.

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9Use of supercritical CO2 for bone delipidation.

FAGES, Jacques., MARTY, Alain., DELGA, Corinne., et al.

Biomaterials, 1994, Vol.15, N°9, p. 650-656.

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