Intelligent scaffolds for tissue engineering of bone, skin and cartilage

A plasma gas method has been used to activate surfaces of porous and non porous polymer and composite materials containing CaP with bioresorbable polymers. This method changes the surface energy and hydrophilic/hydrophobic nature of the materials by the introduction of active groups on the surface. The impact of gas plasma sterilisation has also been conducted on activated surfaces. These materials have been evaluated by tof-Sims and contact angle measurements to assess the activation effect as a function of time. This method will be useful for attaching active components such as antibiotics, either by chemical linking or adsorption.

Two types of surface modification have been performed on the bone scaffolds: (i) the deposition of a biomimetic calcium phosphate (CaP) coatings rendering the scaffold surface osteoconductive and osteoinductive and (ii) gas plasma treatment (Carbon dioxide (CO2), oxygen (O2) or ammoniac (NH3)) known to improve cell attachment on polymers. The biomimetic CaP coating procedure is a two-step aqueous procedure developed previously at IsoTis that allows the deposition of a CaP layer on heat-sensitive or complex-shaped substrates. Therefore this procedure was utilized in the Intelliscaf project on various selected developed bone scaffolds (porous titanium, porous polymers and porous ceramics). Octacalcium phosphate (OCP) coating was homogeneously deposited inside the various porous scaffolds. In vivo, these OCP coated bone scaffolds have shown osteoinductive properties ectopically and an increased osteoconductive potential orthotopically. Gas plasma treatment was used on porous biphasic CaP scaffolds (BCP: a mixture of Hydroxyapatite (HA) and tricalcium phosphate (TCP)) in order to evaluate the cell attachment behaviour. The results did not show considerable effect of the plasma treatment on cell attachment on BCP surfaces.

A porous composite scaffold has been produced with enhanced mechanical properties whilst maintaining interconnections between the pores. The use of polymer allows for the inclusion by adsorption or chemical bonding of active molecules to improve biological response to the scaffolds on implantation. Surface activation has been achieved to modify the hydrophillic/hydrophobic response to the scaffolds. This has been documented using Tof-SIMS and contact angle measurements. Surface activation has also been key in attachment of active molecules e.g. antibiotics for localised release on implantation.

Two in vivo studies in goats, in which various materials for bone regeneration supplied by the partners were tested in both ectopic environment to test osteoinductivity and orthotopic environment to determine bone forming capacity have discriminated two most promising scaffolds: BCP1150 produced by the University of Twente and DTI BCP, i.e. BCP produced by Danish Technological Institute. Both these materials are a biphasic calcium phosphate ceramics composed of tricalcium phosphate (TCP; 5 to 30%) and hydroxyapatite (HA; 95 to 70%) sintered at temperatures between 1150C and 1200C. They were both osteoinductive, i.e. able to induce bone formation intramuscularly and they showed highly pronounced bone forming capacity, which makes them promising candidates for use as synthetic scaffolds for bone repair and regeneration alone or in tissue-engineered constructs. A few in vitro studies have been initiated on various BCP scaffolds with different macro-and microstructures in order to understand the in vivo obtained results, however, most in vitro data were contradictory to the in vivo results.

Gas plasma sterilisation is potentially a good alternative for sterilisation of porous scaffold implant. This method is routinely used for sterilisation of restorable implant. The advantage of this sterilisation method consists in its very low aggressively in regards to the material properties. This method does not reduce the molecular weight of polymeric material such as irradiation method do. Moreover the gas plasma is a quite dry method and low temperature method in comparison to ethylene dioxide sterilisation. All these parameters are of importance for scaffolds which can be made of polymeric materials and can include active substance or cells that can be sensitive to ethylene dioyde or irradiation. On the other hand, gas plasma sterilisation has a poor ability to diffuse in small pores. Most of the scaffolds samples containing PLA's produced in the Intelliscaf project have been sterilised by gas plasma and no contamination problems have been pointed out. This method seems to preserve the material properties of the porous scaffold but sterilisation standard for medical device request a very high sterilisation level. A feasibility study has been conducted in order to evaluate the sterilisation effect on porous scaffold. This initial study has demonstrated that plasma sterilisation works for scaffolds.

A foaming technique has been developed for producing porous Calcium Phosphate (CaP) ceramic scaffolds for bone tissue regeneration. The technique uses as feed stock the CaP ceramic powders including hydroxyapatite, (HA), bi-phasic hydroxyapatite (HA/bTCP) and carbonated hydroxyapatite (CHA). The scaffolds can be produced as granules or machined to the required dimensions. The scaffold production technique can be used for producing porous structures with gradient porosities and can be applied to a wide range of materials including aluminium oxide, zirconium oxide ceramics and metals.

A proprietary spinning method has been developed for producing bioresorbable fibremats for use as scaffolds for tissue regeneration. The mats can be produced from pure bioresorbable polymers or composites of CaP with bioresorbable polymers. Fibre diameters and mat bulk density can be varied. The surfaces of these fibres can be modified at the nano scale.

The relevant scafoolds are made of triblock PLA-PEG-PLA copolymers selected to fulfil as much as possible the criteria imposed by the treament of burns or ulcers by using dermo epidermal constructs obtained by culturing succesively dermal fibroblats in the 3D porous matrix and keratinocytes at the top. The constructs were tested for integration in nude mice. Vascularisation was shown. Data look promising.

Results description: A synthesis process to obtain a Ca/P derivatives powders were optimized: CHA (carbonate-hydroxyapatite. Optimisation of a manufacturing process to obtain reproducible batches of Ca/P (pilot scale). Dissemination and use potential: The devices developed could be used also as a release system for active element for pharmaceutical use. Key innovative feature of the result: Device with controlled porosity: interganular porosity, macroporosity and interconnected porosity. Main expected benefit could be to have a delivery system for bone, local diseases treatment to avoid systemic approach.

Results description: A synthesis process to obtain a Ca/P derivatives powders were optimized: CHA (carbonate-hydroxyapatite) and HA/TCP (biphasic calcium phosphate) and optimisation of a manufacturing process to obtain reproducible batches of Ca/P (pilot scale): manufacturing 3-D bone scaffolds based on CHA and HA Dissemination and use potential: 3-D smart scaffold as bone substitute for different surgical application. Key innovative feature of the result: Synthetic bone graft, no limitation on the quantity and repeatability of the performance. Current status and use of the result and its expected benefits. Strength/Weaknesses/Opportunity/Thread Analysis was carried out: - Competitors analysis. - Potential EU Market Bone graft Substitute analysis. Bone Graft Procedure by Material (2005): 426.200. Autograft 54.7%, Allograft 28.9%, Bone Graft Substitute (BGS) 16.4% penetration. Potential EU Market (Bone Graft Substitutes): the Bone Graft Substitute Market in 2005 was valued at 50 m¬ in Europe with a forecast increase at an average CAGR (Compaund Annual Growth Rate) of 13% from 2005 to 2009. Bone Graft Substitute by Material (2005): DBM 10.6%, Synthetic 49.5% and Xenograft 39.9% penetration. Main expected benefit could be to have a more safety and effective device in respect to other synthetic material and more safe in respect to DBM and Xenograft material.

To date, we have been able to prepare several scaffolds for bone and cartilage regeneration with controlled 3D architecture, composition and microstructure. These scaffolds were developed using various techniques in order to change porosity, interconnectivity, mechanical strength and surface chemistry. Compression moulding and tri-dimensional (3D)-fiber deposition of various compositions of PEOT-PBT copolymer blocks (PolyactiveTM) have been used in order to generate polymer scaffold for cartilage regeneration with various architecture (porosity, interconnectivity), mechanical properties and surface chemistry. Titanium (Ti), calcium-phosphates (CaP) and PolyactiveTM have been used as base materials to produce scaffolds for bone repair with controlled architecture and chemical composition. Porous Ti bone scaffolds were generated by a positive replica method, while CaP ceramic bone scaffolds were prepared out of a CaP slurry containing porogens and subsequent sintering. Porous polymer bone scaffolds were produced by compression moulding. All the developed scaffolds have shown some improved features as compared to the existing scaffolds and will be potentially relevant in use as implants in orthopaedic and cranio-facial surgery. Further test are needed to investigate their clinical applicability.

This process applies a known protocol that was adapted to the case of bioresourcable PLA polymers and feasible for bone tissue reconstruction. Presently, the process is exploitable at lab scale. Scaling-up has to be investigated. The porous structures are adapted to cell seeding and culture.

Carboxyl chain ends of PLA50 oligomers of PLA are activated by thionyl chloride in an organic solvent. The resulting acyl chloride functions can be allowed to react to Labile hydrogen-bearing molecules for the sake of coupling. The method was applied to various compounds including an antibiotic drug, namely amoxicillin.

1) Lyophilisation The lyophilisation processes are described in literature. In respect to PGLA-co-polymers a dioxane solvent route is pursued and in the case of natural polymers a traditional route with water and a co-solvent is used. The results in respect to the lyophilisation process lie in the topics of understanding freezing (for pore size control) and drying dynamics (for stability), concentration dependence (softness and flexibility) and characterization procedures of the resulting scaffolds. 2) Fibre spinning and woven/ non-wovens Electro-spinning is a well-known fibre spinning process for scaffolds for soft tissue. But this is rather expensive and a slow process for commercially application. Therefore two different processes have been developing for fibre production. A wet spinning process for natural water soluble polymers and a melt spinning for thermoplastic synthetic biopolymers. Fibre diameter is not in nano scale but in the lower micrometer fibre (< 10my). In vitro studies have showed that cells like fibroblasts and kerationcytes prepare fibre diameters close to 10 micron. 3) Scaffold prepared by lyophilisation and fibre spinning have been tested both in vitro and in vivo. Both types of scaffolds show attachment and in-growth of skin cells like fibroblasts and kerationcytes. 4) Market studies: The wound care market may be described in various ways. In respect to types of wounds it could be ranked according to economic size of segments as follows. Chronic wounds: leg ulcers, diabetic ulcers, pressure sores, cancer wounds and acute wounds: Traumatic wounds, surgical wounds, and burns. Another way of describing the wound care area is through technologies. Figures in EURO (realized market): Acute wounds: 2.5 bill and 2.5 bill Chronic wounds. The products of the project aim for an advance dressing for the chronic wound care market. The avanced products may be doped with living cells, growth factors and structural elements associated with normal healing. Dermagraft (Smith and Nephew) is an example of a product holding living fibroblasts to a price level of in the range of 25 EURO/ sqcm ($2000 for 10 x 10 cm). Integra (Johnson & Johnson) is an example of a a-cellular bio-product priced in the range of 5.5 EURO/ sqcm ($ 550 for 10 x 12.5 cm). It is the strategy of the project to price the developed product considerable below these price levels probably in the range of 2 to 3 EURO/ sqcm depending on the proven clinical benefit. 5) Protection of results: Coloplast has the policy of making an active effort of identifying inventions that may be protected by patents and if so secure the expedite submission of a patent application. 6) Freedom to operate. The project operates in a field where there already exist substantial amounts of prior art. This will be respected by continuous updating a search profile in patent databases. In respect to freedom to operate it is important that the field also appears to be heavily loaded with publications. 7) Exploitation and dissemination strategy. The dissemination of results and conclusion will predominantly be disclosed through potential use in scaffolds for soft tissue repair. However, the findings may be of relevance to other groups of the consortium as well and is revealed during the project period to our partners. A first product based on the developed technology is expected to reach the market in 2009 through Coloplast's own sales organization or in collaboration with a partner. A detailed business plan will be developed when a thorough understanding of the properties of the final product are established.

Synthesis and manufacturing of PLA 98 setreocopolymer dedicated for injection moulding of resobable orthopaedic fixation device is well controlled in Phusis compagny. This material demonstrates mechanical performance adapted to these standard surgical devices. In the case of scaffold implants, a faster resorption kinetic is required in order to adapt the resorption of the polymer with the kinetic of tissue integration. PLA 50 has been selected as a potential candidate for scaffold production. Manufacturing of large quantities of PLA 50 has already been performed in the company but in small quantities for laboratories research. A manufacturing process of larger quantities of PLA 50 with a controlled molecular weight has been developed during the Intelliscaf project.

A method has been developed for producing highly porous 3D scaffolds of carbonated hydroxyapatite with controlled crystallinity suitable for use as a bone tissue substitute for tissue regeneration. Relevant surgical applications include orthopaedic, maxillofacial, spine surgery etc. The process can be adapted and optimised for use with commercially available carbonated hydroxyapatite powders. Of particular interest is the control of the sintering process using a dedicated prototype furnace for controlling the degree of carbonation?

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