Properties and modification of porous 3-D collagen/hydroxyapatite composites

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Abstract

A freeze drying technique was used to form porous three-dimensional collagen matrixes modified by the addition of a variable amount of nano-hydroxyapatite. For chemical cross-linking EDC/NHS were used. Physical cross-linking was achieved by dehydrothermal treatment. Mechanical properties, morphology, dissolution, porosity, density, enzymatic degradation and swelling properties of materials have been studied after cross-linking. The density of scaffolds and its compressive modulus increased with an increasing amount of hydroxyapatite and collagen concentration in the composite scaffold, while the swelling ratio and porosity decreased. The studied scaffolds dissolved slowly in PBS solution. DHT cross-linked collagen matrices showed a much faster degradation rate after exposure to collagenase than the EDC cross-linked samples.

Introduction

Biomaterials play a very important role in tissue engineering. Numerous scaffolds produced from a variety of biomaterials have been used in biomedical field in attempts to regenerate different tissue and organs in the body [1]. An ideal scaffold should have the following characteristics: biocompatibility, non toxicity, suitable mechanical properties and a biodegradation rate that matches the rate of tissue regeneration. In addition, the biodegraded product should not have negative effects on the surrounding tissues and organs [1], [2], [3]. The architecture of scaffolds used for tissue engineering is of critical importance. Scaffolds should have high porosity and an interconnected porous structure to provide adequate space for the cell's seeding, growth and proliferation [1], [2], [4].

Many methods have been developed to prepare porous three-dimensional biodegradable scaffolds for tissue engineering, including freeze-drying, gas-forming foam, three-dimensional printing, thermal-induced phase separation, electrospinning [5], [6], laser treatment [7] and precipitation of crystals [8], [9].

Collagen is an especially abundant protein in animals. It is the main protein of connective tissue and the main component of the skin. As an extracellular matrix protein it is widely used as a biomaterial for tissue regeneration and implantation. Bone and teeth are both made of collagen with the addition of mineral crystals, mainly hydroxyapatite. Collagen-based materials are widely used in reconstructive medicine. Various applications of collagen in tissue regeneration may include: artificial skin, bone graft substitutes, dental implants, implants for incontinence, artificial tendons and blood vessels, corneal implants, nerve regeneration, regeneration of cartilage, regeneration of skin and organs. However, soluble collagen can be extracted only from very young tissue. This fact makes soluble collagen very expensive and rather rare material [10]. Scientific reports indicate that they are several research groups working on preparation of appropriate materials for ideal scaffold. In particular the collagen based materials with sufficient mechanical parameters which could be used as artificial bone are of the huge interest [2], [3], [4], [5], [6], [7], [8], [9], [10].

The disadvantage of using collagen as a biomaterial for tissue repair is its high degradation rate, which leads rapidly to a loss of mechanical properties [2]. Many attempts have been made to overcome this problem through the means of mixing collagen with either natural (e.g. elastin [11], [12], chitosan [10], [12], glycoaminoglycans – GAGs [13], [14]) or synthetic polymers (e.g. poly(vinyl alcohol) – PVA [15], polycaprolactone – PCL [6], [16], polylactic acid – PLLA [6], polyglycolic acid – PGA [6], [17]) or by adding mineral crystals [18], [19]. Likewise, the physical properties of collagen biomaterials are improved by applying different cross-linking method. Different chemical and physical cross-linking methods are used for crosslinking of protein materials. The physical cross-linking agent such as gamma radiation, UV-irradiation, heat and dehydrothermal treatment can be used [6], [20]. However, the energy of UV and γ-radiation can destroy the native structure of the protein. In DHT, cross-linking is induced by heating dry collagen under vacuum to ∼100 °C. The DHT technique induces an increase in tensile strength and some fragmentation in the collagen's molecular structure [20]. Chemical cross-linking can produce highly cross-linked material in a very short time. There are several chemical compounds capable of cross-linking proteins, including glutaraldehyde, formaldehyde, polyepoxy compounds, acyl azide, carbodiimides and hexamethylene-diisicyanate [6], [20], [21]. Anyway, these treatments are not sufficiently cytocompatible due to the potential toxicity of some of the cross-linking agents utilized. Covalent cross-linking using EDC/NHS {N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)} is a widely used method in biomaterials preparations [20], [22], [23], [24].

The aim of this study was to determine the optimal content of HAp in porous collagen matrices and to study the chemical and physical cross-linking of composite samples with different collagen–hydroxyapatite ratios. The samples were treated using EDC/NHS. Collagen–hydroxyapatite samples have been also crosslinked using temperature to compare chemical cross-linking with physical cross-linking. Such materials can be applied as a scaffold for bone tissue restoration. To clarify the effects of EDC/NHS and DHT cross-linking the following properties were measured: water uptake ability, dissolution, enzymatic degradation, porosity, density and mechanical properties.

Section snippets

Materials

Collagen (Col) was obtained in our laboratory from the tail tendons of young rats (Fig. 1). Hydroxyapatite (HAp) (nanopowder, <200 nm particle size), N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were supplied by the company Sigma–Aldrich (Poland).

Scaffold preparation

The high porous scaffolds were produced from a collagen–hydroxyapatite suspension using a freeze-drying technique. First, collagen solutions with concentrations of 1% and 2% (w/w) were prepared from

Scaffold morphology

The porous collagen matrices with different HAp contents were fabricated by freeze-drying and cross-linked using EDC/NHS or DHT. Porosity is the key parameter for the scaffold design. A highly porous scaffold plays a critical role in cell seeding, proliferation and new tissue formation in three dimensions (3D). An ideal bone scaffold should have sufficient porosity (such as ≥90%) to accommodate osteoblasts or osteoprogenitor cells [2], [25]. Scanning electron microscopy (SEM) was used to

Conclusions

Highly porous collagen–hydroxyapatite scaffolds were produced in this study. The collagen/hydroxyapatite scaffolds showed good swelling properties dependent on the ratio of the components. The rate of degradation, microstructure, mechanical and swelling properties of the composite scaffold can be modified by the cross-linking method and by changing the collagen concentration or through the addition of hydroxyapatite.

EDC/NHS as cross-linking agent leads to an increase of mechanical properties of

Acknowledgment

Financial support from the Ministry of Science (MNiSW, Poland) Grant No N N507 349535 and COST Action TD 0903 EU is gratefully acknowledged.

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